Spray freeze-dried compositions

ABSTRACT

A process for producing a powder comprises spray freeze-drying an aqueous solution or suspension comprising a pharmaceutical agent, said solution or suspension having a solids content of 20% by weight or more. The spray freeze-dried powder may be administered to a subject via a needleless syringe.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to U.S. provisional patentapplication serial No. 60/296,939, filed Jun. 8, 2001, from whichapplication priority is claimed pursuant to 35 U.S.C. §119(e)(1) andwhich application is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to processes for producing pharmaceuticalcompositions which are suitable for transdermal particle delivery from aneedleless syringe system.

BACKGROUND OF THE INVENTION

[0003] The ability to deliver pharmaceutical agents into and throughskin surfaces (transdermal delivery) provides many advantages over oralor parenteral delivery techniques. In particular, transdermal deliveryprovides a safe, convenient and noninvasive alternative to traditionaladministration systems, conveniently avoiding the major problemsassociated with oral delivery (e.g. variable rates of absorption andmetabolism, gastrointestinal irritation and/or bitter or unpleasant drugtastes) or parenteral delivery (e.g. needle pain, the risk ofintroducing infection to treated individuals, the risk of contaminationor infection of health care workers caused by accidental needle-sticksand the disposal of used needles).

[0004] However, despite its clear advantages, transdermal deliverypresents a number of its own inherent logistical problems. Passivedelivery through intact skin necessarily entails the transport ofmolecules through a number of structurally different tissues, includingthe stratum corneum, the viable epidermis, the papillary dermis and thecapillary walls in order for the drug to gain entry into the blood orlymph system. Transdermal delivery systems must therefore be able toovercome the various resistances presented by each type of tissue.

[0005] In light of the above, a number of alternatives to passivetransdermal delivery have been developed. These alternatives include theuse of skin penetration enhancing agents, or “permeation enhancers,” toincrease skin permeability, as well as non-chemical modes such as theuse of iontophoresis, electroporation or ultrasound. However, thesealternative techniques often give rise to their own unique side effectssuch as skin irritation or sensitization. Thus, the spectrum of agentsthat can be safely and effectively administered using traditionaltransdermal delivery methods has remained limited.

[0006] More recently, a novel transdermal drug delivery system thatentails the use of a needleless syringe to fire powders (i.e., soliddrug-containing particles) in controlled doses into and through intactskin has been described. In particular, commonly owned U.S. Pat. No.5,630,796 to Bellhouse et al. describes a needleless syringe thatdelivers pharmaceutical particles entrained in a supersonic gas flow.The needleless syringe is used for transdermal delivery of powdered drugcompounds and compositions, for delivery of genetic material into livingcells (e.g., gene therapy) and for the delivery of biopharmaceuticals toskin, muscle, blood or lymph. The needleless syringe can also be used inconjunction with surgery to deliver drugs and biologics to organsurfaces, solid tumors and/or to surgical cavities (e.g., tumor beds orcavities after tumor resection). In theory, practically anypharmaceutical agent that can be prepared in a substantially solid,particulate form can be safely and easily delivered using such devices.

[0007] To enable powdered drug compositions to be effectivelyadministered via this new needleless syringe technique, the powdersshould have certain physical characteristics. In particular, the size ofthe particles which form the powders should be controllable, preferablywith a narrow size distribution. Further, the particle density should behigh, the particles should be free-flowing under a dry environment andtheir moisture content should be low. Additional properties of theparticles which are desired include a spherical shape and a smoothsurface. Each of these properties is important to provide good skinpenetration whilst avoiding damage to the particles themselves under theforces required for delivery via needleless syringe.

[0008] One of the most important factors in determining the physicalcharacteristics of the powders is the particular manner by which theyare produced. Various spray freeze-drying techniques have previouslybeen described for, for example, the preparation of powders for aerosoldelivery and microspheres for conventional drug delivery. In theseapplications, particles are desired to be light and porous, propertieswhich are inherent in powders produced by previously described sprayfreeze-drying techniques. Such light and porous particles are notsuitable for use in needleless syringe devices.

[0009] Maa et al. (1999) Pharmaceuticals Research 16(2) describe thephysical characteristics of spray-freeze-dried particles and theirperformance as aerosols. The spray freeze-drying process is said torender highly porous particles with a large specific surface area. Maaestimated that the particle density of spray freeze-dried particles istypically approximately one ninth of that of equivalent particles driedby spray-drying.

[0010] U.S. Pat. No. 5,019,400 describes a process for preparingmicrospheres using very cold temperatures to freeze polymer-biologicallyactive agent mixtures into polymeric microspheres with retention ofbiological activity and material. Polymer is dissolved in a solventtogether with an active agent that can be either dissolved in thesolvent or dispersed in the solvent in the form of microparticles. Thepolymer/active agent mixture is atomised into a vessel containing aliquid non-solvent, alone or frozen and overlayed with a liquified gas,at a temperature below the freezing point of the polymer/active agentsolution.

[0011] When the combination with the liquified gas is used, the atomiseddroplets freeze into microspheres upon contacting the cold liquifiedgas, then sink onto the frozen non-solvent layer. The frozen non-solventis then thawed. As the non-solvent thaws, the microspheres which arestill frozen sink into the liquid non-solvent. The solvent in themicrospheres then thaws and is slowly extracted into the non-solvent,resulting in hardened microspheres containing active agent either as ahomogeneous mixture of the polymer and the active agent or as aheterogeneous two phase system of discrete zones of polymer and activeagent.

[0012] If a cold solvent is used alone, the atomized droplets freezeupon contacting the solvent, and sink to the bottom of the vessel. Asthe non-solvent for the polymer is warmed, the solvent in themicrospheres thaws and is extracted into the non-solvent, resulting inhardened microspheres.

SUMMARY OF THE INVENTION

[0013] The present inventors have surprisingly found that sprayfreeze-drying a solution or suspension having a high solids content inthe solvent produces particles which are quite dense and which performwell in needleless syringe devices. The use of a high solids contentstarting material minimises the pore formation which occurs during thedrying step. Sublimation of frozen solvent away from the particlesduring drying is the typical cause of pore formation. Maximising thesolids content in the solution or suspension, and therefore in thefrozen particles, reduces the number and size of pores which form ondrying, thus providing denser particles.

[0014] The inventors have also found that the use of particularexcipients may aid the formation of dense particles. Appropriateexcipient compositions allow particles to collapse and densify duringfreeze-drying. It is also thought that selecting specific excipientcompositions may assist particle formation by increasing the solubilityof certain guest substances (such as peptides and proteins) in thesolvent system. This further aids in maximising the solids content ofthe solution or suspension.

[0015] The present invention therefore enables the spray freeze-dryingprocess, with its attendant advantages, to be adapted to needlelesssyringe requirements. The particles of the invention have a well-definedsize and a high density, together with other mechanical properties whichcollectively are suitable for transdermal delivery via a needlelesssyringe. The present spray freeze-drying process is a simple techniquewhich is highly suitable for scaling-up to commercial production levels.In addition, it has, as yet, been found to be entirely formulationindependent. The technique can therefore be applied to almost anypharmaceutical formulation, a factor which further adds to thecommercial viability of the present invention.

[0016] Accordingly, the present invention provides a process for thepreparation of a powder, which process comprises the step of sprayfreeze-drying an aqueous solution or suspension comprising apharmaceutical agent, said solution or suspension having a solidscontent of 20% by weight or more.

[0017] The invention further provides:

[0018] a dosage receptacle for a needleless syringe, said receptaclecontaining an effective amount of a powder prepared by the process ofthe invention;

[0019] a needleless syringe which is loaded with a powder prepared bythe process of the invention;

[0020] a vaccine composition comprising a pharmaceutically acceptablecarrier or diluent and a powder prepared by the process of theinvention;

[0021] a method of vaccinating a subject, which method comprisesadministering to the said subject an effective amount of a powderprepared by the process of the invention; and

[0022] a powder which is prepared by the process of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIGS. 1a-1 d are optical micrographs (at X100 magnification) ofselected particle formulations assessed in the study described inExample 1 at subpart 1.5.2. FIG. 1a shows particles from Formulation138-20-4A, prepared by spray freeze-drying (SFD) 20 μg of the HBsAgantigen. FIG. 1b shows particles from Formulation 138-16-1C, prepared bySFD and then sieving to obtain a 38-53 μm particle fraction formed from20 μg of HBsAg antigen and 50 μg of alum adjuvant. FIG. 1c showsparticles from Formulation 138-20-4B, prepared by SFD and thencompressing, grinding and sieving (C/G/S) to obtain a 38-53 μm particlefraction formed from 20 μg of HBsAg antigen. FIG. 1d shows particlesfrom Formulation 138-20-5C, prepared by SFD and then C/G/S to obtain a38-53 μm particle fraction formed from 20 μg of HBsAg antigen and 50 μgof alum adjuvant.

[0024]FIGS. 2a-2 d are SEM micrographs of selected particle formulationsassessed in the study described in Example 1 at subpart 1.5.3. FIG. 2ashows particles from Formulation 138-20-4A, prepared by SFD 20 μg of theHBsAg antigen. FIG. 2b shows particles from Formulation 138-16-1C,prepared by SFD and then sieving to obtain a 38-53 μm particle fractionformed from 20 μg of HBsAg antigen and 50 μg of alum adjuvant. FIG. 2cshows particles from Formulation 138-20-4B, prepared by SFD and thenC/G/S to obtain a 38-53 μm particle fraction formed from 20 μg of HBsAgantigen. FIG. 2d shows particles from Formulation 138-20-5C, prepared bySFD and then C/G/S to obtain a 38-53 μm particle fraction formed from 20μg of HBsAg antigen and 50 μg of alum adjuvant.

[0025]FIGS. 3a and 3 b are optical images of liquid suspensions of twoalum-containing formulations assessed in the study described in Example1 at subpart 1.5.7. FIG. 3a is an optical image of the reconstitutedformulation 138-16-1 prepared by SFD 20 μg of the HBsAg antigen. FIG. 3bis an optical image of the reconstituted formulation 138-20-5C preparedby SFD and then C/G/S to obtain a 38-53 μm particle fraction formed from20 μg of HBsAg antigen and 50 μg of alum adjuvant.

[0026]FIGS. 4a and 4 b are photos of the non-reducing SDS-PAGE gel (FIG.4a) and reducing SDS-PAGE gel (FIG. 4b) showing the results of the studydescribed in Example 1 at subpart 1.5.8.

[0027]FIG. 5 is a schematic of the Spray Freeze-Drying (SFD) processdescribed in Example 2 at subpart 2.3.1.

[0028]FIGS. 6a-6 d are optical micrographs (at X100 magnification) ofselected particle formulations assessed in the study described inExample 2 at subpart 2.5.1. FIG. 6a shows particles from Formulation156-16-1 prepared by SFD a composition containing 10% BSA, 45%trehalose, 27% mannitol, 18% PVP (K17) and 0.1% Pluronic F68. FIG. 6bshows particles from Formulation 156-16-2 prepared by SFD a compositioncontaining 10% BSA, 44.9% trehalose, 26.9% mannitol, 18% PVP (K17), 0.1%methionine, and 0.1% Pluronic F68. FIG. 6c shows particles fromFormulation 156-16-3 prepared by SFD a composition containing 10% BSA,26.9% trehalose, 26.9% mannitol, 35.9% PVP (K17), 0.1% methionine, and0.1% Pluronic F68. FIG. 6e shows particles from Formulation 156-16-4prepared by SFD a composition containing 10% BSA, 26.9% trehalose, 26.9%mannitol, 35.9% PVP (K17), 0.1% methionine, and 0.1% Pluronic F68.

[0029]FIGS. 7a-7 f are optical micrographs (at X100 magnification) ofselected particle formulations assessed in the study described inExample 2 at subpart 2.5.2. FIG. 7a shows particles from Formulation156-35-1 prepared by SFD a composition containing 10% BSA, 36%raffinose, 27% trehalose, and 27% mannitol. FIG. 7b shows particles fromFormulation 156-35-2 prepared by SFD a composition containing 10% BSA,36% raffinose, 36% mannitol, and 18% PVP (K17). FIG. 7c shows particlesfrom Formulation 156-35-3 prepared by SFD a composition containing 10%BSA, 40% raffinose and 30% mannitol. FIG. 7d shows particles fromFormulation 156-42-1 prepared by SFD a composition containing 10% BSA,36% raffinose, 27% trehalose, and 27% mannitol. FIG. 7e shows particlesfrom Formulation 156-42-2 prepared by SFD a composition containing 10%BSA, 27% raffinose, 27% mannitol, 18% glycine and 18% trehalose. FIG. 7fshows particles from Formulation 156-42-4 prepared by SFD a compositioncontaining 10% BSA, 27% raffinose, 27% sucrose, and 36% mannitol FIGS.8a-8 f are optical micrographs (at X100 magnification) of selectedparticle formulations assessed in the study described in Example 2 atsubpart 2.5.3. FIG. 8a shows particles from Formulation 156-35-4prepared by SFD a composition containing 10% BSA, 27% trehalose, 27%mannitol and 36% dextran (10 lkDa). FIG. 8b shows particles fromFormulation 156-42-3-1 prepared by SFD a composition containing 10% BSA,27% trehalose, 27% mannitol and 36% dextran (10 kDa). FIG. 8c showsparticles from Formulation 156-42-3-2 prepared by SFD a compositioncontaining 10% BSA, 27% trehalose, 27% mannitol and 36% dextran (10kDa). FIG. 8d shows particles from Formulation 156-53-1 prepared by SFDa composition containing 10% BSA, 27% trehalose, 27% mannitol and 36%dextran (10 kDa). FIG. 8e shows particles from Formulation 156-61-1prepared by SFD a composition containing 10% BSA, 27% trehalose, 27%mannitol and 36% dextran (10 kDa). FIG. 8f shows particles fromFormulation 156-65-1 prepared by SFD a composition containing 10% BSA,36% trehalose, 18% mannitol, 18% arginine glutamate, and 18% dextran (10kDa).

[0030]FIGS. 9a-9 f are optical micrographs (at X100 magnification) ofselected particle formulations assessed in the study described inExample 2 at subpart 2.5.4. FIG. 9a shows particles from Formulation156-57-1 prepared by SFD a composition containing 10% BSA, 36%trehalose, 36% mannitol, and 18% alanine. FIG. 9b shows particles fromFormulation 156-57-2 prepared by SFD a composition containing 10% BSA,27% trehalose, 27% mannitol, and 36% arginine glutamate. FIG. 9c showsparticles from Formulation 156-65-2 prepared by SFD a compositioncontaining 10% BSA, 36% trehalose, 18% mannitol, and 36% arganineglutamate. FIG. 9d shows particles from Formulation 156-71-1 prepared bySFD a composition containing 10% BSA, 36% trehalose, 18% mannitol, and36% arganine glutamate. FIG. 9e shows particles from Formulation156-76-1 prepared by SFD a composition containing 10% BSA, 35.9%trehalose, 18% mannitol, 35.9% arginine glutamate, 0.1% Pluronic F168,and 0.1% methionine. FIG. 9f shows particles from Formulation 156-76-2prepared by SFD a composition containing 10% BSA, 26.9% trehalose, 26.9%mannitol, 35.9% arginine glutamate, 0.1% Pluronic F168, and 0.1 %methionine.

[0031]FIGS. 10a-10 c are optical micrographs (at X100 magnification) ofselected particle formulations assessed in the study described inExample 2 at subpart 2.5.5. FIG. 10a shows particles from Formulation156-80-1 prepared by SFD a composition containing 10% BSA, 27%trehalose, 27% mannitol, and 36% arginine aspartate. FIG. 10b showsparticles from Formulation 156-80-2 prepared by SFD a compositioncontaining 10% BSA, 5% Pluronic F168, 59.5% trehalose, and 25.5%mannitol. FIG. 10c shows particles from Formulation 156-80-3 prepared bySFD a composition containing 10% BSA, 35.9% trehalose, 18% mannitol,35.9% arginine glutamate, 0.1% methionine and 0.1% Tween 80.

[0032]FIGS. 11a-11 d are optical micrographs of selected particleformulations assessed in the study described in Example 3 at subpart3.4. FIG. 11a shows the Adju-Phos adjuvant (2 w/v % placebo AlPO₄) gelafter freezing at −20° C. and thawing under ambient conditions. FIG. 11bshows the Adju-Phos gel after spray-freezing and then thawing. FIG. 11cshows the Alhydrogel adjuvant (2 w/v % placebo Al(OH)₃) gel afterfreezing at −20° C. and thawing at ambient conditions. FIG. 11d showsthe Alhydrogel gel after spray-freezing and then thawing.

[0033]FIGS. 12a and 12 b depict the particle size analysis described inExample 3, subpart 3.5.1. FIG. 12a shows the particle size analyses byAccuSizer for: (a) a starting alum-adsorbed HBsAg gel, depicted by the(♦) curve on the graph; (b) a “high Alum SFD” composition formed fromAlum hydroxide (3.0 w/v %), mannitol (1.9 w/v %), glycine (0.5 w/v %),and dextran (0.61 w/v %), depicted by the (⋄) curve on the graph; and(c) a “low Alum SFD” composition formed from Alum hydroxide (0.6 w/v %),mannitol (2.8 w/v %), glycine (1.2 w/v %), and dextran (0.58 w/v %),depicted by the (*) curve on the graph. FIG. 12b shows the particle sizeanalyses by AccuSizer for: (a) a starting alum-adsorbed HBsAg gel,depicted by the (♦) curve on the graph; and a freeze dried, C/G/Sprocessed composition formed from Alum hydroxide (3.0 w/v %), mannitol(1.9 w/v %), glycine (0.5 w/v %), and dextran (0.61 w/v %), depicted bythe (*) curve on the graph.

[0034]FIG. 13 shows the ELISA results obtained in Example 3, subpart3.5.1, reported as anti-HBsAg antibody responses elicited in theimmunized animals receiving “FD” (SFD composition formed from Alumhydroxide (3.0 w/v %), mannitol (1.9 w/v %), glycine (0.5 w/v %), anddextran (0.61 w/v %)); “FD <20 μm” (SFD composition formed from Alumhydroxide (3.0 w/v %), mannitol (1.9 w/v %), glycine (0.5 w/v %), anddextran (0.61 w/v %), particle size 20μm fraction); “FD 38-53 μm” (SFDcomposition formed from Alum hydroxide (3.0 w/v %), mannitol (1.9 w/v%), glycine (0.5 w/v %), and dextran (0.61 w/v %), particle size 38-53μmfraction); “FD 53-75μm” (SFD composition formed from Alum hydroxide (3.0w/v %), mannitol (1.9 w/v %), glycine (0.5 w/v %), and dextran (0.61 w/v%), particle size 53-78μm fraction); “SFD 3% Alum” (SFD compositionformed from Alum hydroxide (3.0 w/v %), mannitol (1.9 w/v %), glycine(0.5 w/v %), and dextran (0.61 w/v %)); “SFD 0.6% Alum” (SFD compositionformed from Alum hydroxide (0.6 w/v %), mannitol (2.8 w/v %), glycine(1.2 w/v %), and dextran (0.58 w/v %)); and “untreated” the untreatedcontrol animals.

[0035]FIGS. 14a-14 b show the ELISA results obtained in Example 3,subpart 3.5.2, reported as anti-dT (FIG. 14a) and anti-tT (FIG. 14b)antibody responses elicited in the immunized animals receiving either:(a) the “SFD” composition formed from alum phosphate (1.5 w/v %),trehalose (1.5 w/v %), glycine (0.4 w.v %), and dextran (0.6 w/v %),delivered by epidermal powder injection (“EPI”); (b) the “SD”composition formed from alum phosphate (5 w/v %) and trehalose (5 w/v%), delivered by EPI; or (c) the “untreated” composition which was aliquid DT vaccine composition administered by intramuscular (IM)injection.

[0036]FIGS. 15a-15 e are digital light microscope images of H&E stainedsections from the histological skin samples taken in the study describedin Example 4, subpart 4.4.1, and showing histological changes in theimmunization sites. FIG. 15a shows normal skin (20X); FIG. 15b shows thesite of EPI administration (24X); FIG. 15 c shows the site of EPIadministration (105X); FIG. 15d shows the site of intradermal (ID)injection (20X) and FIG. 15e shows the site of ID injection (105X).

[0037]FIGS. 16a and 16 b are scanning electron micrographs (SEMs) of SFDalum powder formulations described in Example 4 at subpart 4.5.1. Theformulations were formed from compositions containing trehalose (30%),mannitol (30%), dextran (40%) at 35 w/w % of total solid content. FIG.16a shows an aluminium hydroxide composition, and FIG. 16b shows analuminium phosphate composition.

[0038]FIGS. 17a- 17 d are optical micrographs assessing alum coagulationin selected particle formulations as described in Example 4 at subpart4.4.1. FIG. 17a shows a reconstituted freeze dried (FD) alum hydroxidecomposition formulated with trehalose (30%), mannitol (30%), dextran(40%) at 35 w/w % of total solid content. FIG. 17b shows a reconstitutedSFD alum hydroxide composition formulated with trehalose (30%), mannitol(30%), dextran (40%) at 35 w/w % of total solid content. FIG. 17c showsa reconstituted FD alum phosphate composition formulated with trehalose(30%), mannitol (30%), dextran (40%) at 35 w/w % of total solid content.FIG. 17d shows a reconstituted SFD alum phosphate composition formulatedwith trehalose (30%), mannitol (30%), dextran (40%) at 35 w/w % of totalsolid content

[0039]FIGS. 18a-18 b are optical micrographs assessing alum coagulationin selected particle formulations as described in Example 4 at subpart4.4.3. FIG. 18a shows a reconstituted SFD alum phosphate compositionformulated with trehalose (30%), mannitol (30%), dextran (40%) andpolysorbate 80 at 0.5 w/w % of total solid content, where the startingtotal solid content was 35 w/w %. FIG. 18b shows a reconstituted SFDalum phosphate composition formulated with trehalose (30%), mannitol(30%), dextran (40%) and polysorbate 80 at 0.5 w/w % of total solidcontent, where the starting total solid content was 40 w/w %. FIG. 18cshows a reconstituted SFD alum phosphate composition formulated withtrehalose (30%), mannitol (30%), dextran (40%) and polysorbate 80 at 0.5w/w % of total solid content, where the starting total solid content was30 w/w %. FIG. 18d shows a reconstituted SFD alum phosphate compositionformulated with trehalose (30%), mannitol (30%), dextran (40%) andpolysorbate 80 at 0.5 w/w % of total solid content, where the startingtotal solid content was 25 w/w %.

[0040]FIGS. 19a and 19 b show the ELISA results obtained in Example 4,subpart 4.6.1, reported as geometric mean anti-HBsAg IgG antibody titersof animals vaccinated with HBsAg vaccine compositions. FIG. 19a showstiters from animals receiving SFD compositions delivered by EPI(“SFD/EPI”), where the compositions were either a SFD formulationcontaining 1 μg HBsAg adsorbed to 25 μg aluminium hydroxide (“Al(OH)₃”)or a SFD formulation containing 1 μg HBsAg adsorbed to 25 μg aluminiumphosphate (“AlPO₄”); animals reciving SFD compositions that werereconstituted to liquid form and delivered via IM injection(“SFD/reconstituted/IM”), where the compositions were either a SFDformulation containing 1 μg HBsAg adsorbed to 25 μg aluminium hydroxide(“Al(OH)₃”) or a SFD formulation containing 1 μg HBsAg adsorbed to 25 μgaluminium phosphate (“AlPO₄”); or a control receiving the commercialHepatitis B vaccine composition containing 1 μg HBsAg adsorbed to 25 μgAl(OH)₃. Titers are reported from week 4, 6 and 9 sera. FIG. 19b showstiters from animals receiving either a reconstituted SFD vaccineformulation containing 2 μg HBsAg adsorbed with 2.5 μg AlPO₄(“SFD/reconstituted”); or a liquid injection of a conventional IMformulation containing 2 μg HBsAg adsorbed to 50 μg AlPO₄(“liquid/commercial”). Titers are reported from weeks 4 and 6 sera.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified compositions or process parameters. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

[0042] All publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

[0043] It must be noted that, as used in this specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to “a particle” includes a mixture of two or moresuch particles, reference to “an excipient ” includes mixtures of two ormore such excipients, and the like.

[0044] A. Definitions

[0045] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although a number ofmethods and materials similar or equivalent to those described hereincan be used in the practice of the present invention, the preferredmaterials and methods are described herein.

[0046] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0047] As used herein, the term “pharmaceutical” or “pharmaceuticalagent” intends any compound or composition of matter which, whenadministered to an organism (human or animal) induces a desiredpharmacologic and/or physiologic effect by local and/or systemic action.The term therefore encompasses those compounds or chemicalstraditionally regarded as drugs, as well as biopharmaceuticals includingmolecules such as peptides, hormones, nucleic acids, gene constructs andthe like. More particularly, the term “pharmaceutical” or“pharmaceutical agent” includes compounds or compositions for use in allof the major therapeutic areas including, but not limited to,anti-infectives such as antibiotics and antiviral agents; analgesics andanalgesic combinations; local and general anaesthetics; anorexics;antiarthritics; antiasthmatic agents; anticonvulsants; antidepressants;antihistamines; anti-inflammatory agents; antinauseants;antineoplastics; antipruritics; antipsychotics; antipyretics;antispasmodics; cardiovascular preparations (including calcium channelblockers, ACE-inhibitors, beta-blockers, beta-agonists andantiarrythmics); antihypertensives; diuretics; vasodilators; centralnervous system stimulants; cough and cold preparations; decongestants;diagnostics; hormones; bone growth stimulants and bone resorptioninhibitors; immunosuppressives; muscle relaxants; psychostimulants;sedatives; tranquilizers; therapeutic proteins (e.g., antigens,antibodies, growth factors, cytokines, interleukins, lymphokines,interferons, enzymes, etc.), peptides and fragments thereof (whethernaturally occurring, chemically synthesized or recombinantly produced);and nucleic acid molecules (polymeric forms of two or more nucleotides,either ribonucleotides (RNA) or deoxyribonucleotides (DNA) includingboth double- and single-stranded molecules, gene constructs, expressionvectors, antisense molecules and the like).

[0048] By “antigen” is meant a molecule which contains one or moreepitopes that will stimulate a host's immune system to make a cellularantigen-specific immune response or a humoral antibody response. Thus,antigens include polypeptides including antigenic protein fragments,oligosaccharides, polysaccharides and the like. Furthermore, the antigencan be derived from any known virus, bacterium, parasite, plant,protozoan or fungus, and can be a whole organism. The term also includestumor antigens. Similarly, an oligonucleotide or polynucleotide whichexpresses an antigen, such as in DNA immunization applications, is alsoincluded in the definition of an antigen. Synthetic antigens are alsoincluded, for example polyepitopes, flanking epitopes and otherrecombinant or synthetically derived antigens (Bergmann et al (1993)Eur. J Immunol. 23:2777-2781; Bergmann et al. (1996) J. Immunol.157:3242-3249; Suhrbier, A. (1997) Immunol. and Cell Biol. 75:402-408;Gardner et al. (1998) 12^(th) World AIDS Conference, Geneva,Switzerland, Jun. 28-Jul. 3, 1998).

[0049] The above pharmaceuticals or pharmaceutical agents, alone or incombination with other agents, are typically prepared as pharmaceuticalcompositions which can contain one or more added materials such ascarriers, vehicles, and/or excipients. “Carriers,” “vehicles” and“excipients” generally refer to substantially inert materials which arenontoxic and do not interact with other components of the composition ina deleterious manner. These materials can be used to increase the amountof solids in particulate pharmaceutical compositions. Examples ofsuitable carriers include water, silicone, gelatin, waxes, and likematerials. Examples of normally employed “excipients,” includepharmaceutical grades of carbohydrates including monosaccharides,disaccharides, cyclodextrans, and polysaccharides (e.g., dextrose,sucrose, lactose, trehalose, raffinose, mannitol, sorbitol, inositol,dextrans, and maltodextrans); starch; cellulose; salts (e.g. sodium orcalcium phosphates, calcium sulfate, magnesium sulfate); citric acid;tartaric acid; glycine; high molecular weight polyethylene glycols(PEG); polyvinylpyrrolidone (PVP); Pluronics; surfactants; andcombinations thereof. Generally, when carriers and/or excipients areused, they are used in amounts ranging from about 0.1 to 99 wt % of thepharmaceutical composition.

[0050] The term “powder” as used herein refers to a composition thatconsists of substantially solid particles that can be deliveredtransdermally using a needleless syringe device. The particles that makeup the powder can be characterized on the basis of a number ofparameters including, but not limited to, average particle size, averageparticle density, particle morphology (e.g. particle aerodynamic shapeand particle surface characteristics) and particle penetration energy(P.E.).

[0051] The average particle size of the powders according to the presentinvention can vary widely and is generally from 0.1 to 250 μm, forexample from 10 to 100 μm and more typically from 20 to 70 μm. Theaverage particle size of the powder can be measured as a mass meanaerodynamic diameter (MMAD) using conventional techniques such asmicroscopic techniques (where particles are sized directly andindividually rather than grouped statistically), absorption of gases,permeability or time of flight. If desired, automatic particle-sizecounters can be used (e.g. Aerosizer Counter, Coulter Counter, HIAC.Counter, or Gelman Automatic Particle Counter) to ascertain the averageparticle size.

[0052] Actual particle density or “absolute density” can be readilyascertained using known quantification techniques such as heliumpycnometry and the like. Alternatively, envelope (“tap”) densitymeasurements can be used to assess the density of a powder according tothe invention. The envelope density of a powder of the invention isgenerally from 0.5 to 25 g/cm³, preferably from 0.8 to 1.5 g/cm³.

[0053] Envelope density information is particularly useful incharacterizing the density of objects of irregular size and shape.Envelope density is the mass of an object divided by its volume, wherethe volume includes that of its pores and small cavities but excludesinterstitial space. A number of methods of determining envelope densityare known in the art, including wax immersion, mercury displacement,water absorption and apparent specific gravity techniques. A number ofsuitable devices are also available for determining envelope density,for example, the GeoPyc™ Model 1360, available from the MicromeriticsInstrument Corp. The difference between the absolute density andenvelope density of a sample pharmaceutical composition providesinformation about the sample's percentage total porosity and specificpore volume.

[0054] Particle morphology, particularly the aerodynamic shape of aparticle, can be readily assessed using standard light microscopy. It ispreferred that the particles which make up the instant powders have asubstantially spherical or at least substantially elliptical aerodynamicshape. It is also preferred that the particles have an axis ratio of 3or less to avoid the presence of rod- or needle-shaped particles. Thesesame microscopic techniques can also be used to assess the particlesurface characteristics, e.g. the amount and extent of surface voids ordegree of porosity.

[0055] Particle penetration energies can be ascertained using a numberof conventional techniques, for example a metallized film P.E. test. Ametallized film material (e.g. a 125 μm polyester film having a 350 Ålayer of aluminum deposited on a single side) is used as a substrateinto which the powder is fired from a needleless syringe (e.g. theneedleless syringe described in U.S. Pat. No. 5,630,796 to Bellhouse etal) at an initial velocity of about 100 to 3000 m/sec. The metallizedfilm is placed, with the metal-coated side facing upwards, on a suitablesurface.

[0056] A needleless syringe loaded with a powder is placed with itsspacer contacting the film, and then fired. Residual powder is removedfrom the metallized film surface using a suitable solvent. Penetrationenergy is then assessed using a BioRad Model GS-700 imaging densitometerto scan the metallized film, and a personal computer with a SCSIinterface and loaded with MultiAnalyst software (BioRad) and Matlabsoftware (Release 5.1, The MathWorks, Inc.) is used to assess thedensitometer reading. A program is used to process the densitometerscans made using either the transmittance or reflectance method of thedensitometer. The penetration energy of the spray freeze-dried powdersshould be equivalent to, or better than that of reprocessed mannitolparticles of the same size (mannitol particles that are freeze-dried,compressed, ground and sieved according to the methods of commonly ownedInternational Publication No. WO 97/48485, incorporated herein byreference).

[0057] The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably herein and refer to a polymeric form of nucleotides ofany length, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three-dimensional structure, andmay perform any function, known or unknown. Non-limiting examples ofpolynucleotides include a gene, a gene fragment, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers.

[0058] A polynucleotide is typically composed of a specific sequence offour nucleotide bases: adenine (A); cytosine (C); guanine (G); andthymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA).Thus, the term nucleic acid sequence is the alphabetical representationof a polynucleotide molecule. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching.

[0059] A “vector” is capable of transferring nucleic acid sequences totarget cells (e.g., viral vectors, non-viral vectors, particulatecarriers, and liposomes). Typically, “vector construct”, “expressionvector”, and “gene transfer vector”, mean any nucleic acid constructcapable of directing the expression of a gene of interest and which cantransfer gene sequences to target cells. Thus, the term includes cloningand expression vehicles, as well as viral vectors. A “plasmid” is avector in the form of an extrachromosomal genetic element.

[0060] A nucleic acid sequence which “encodes” a selected antigen is anucleic acid molecule which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Forthe purposes of the invention, such nucleic acid sequences can include,but are not limited to, CDNA from viral, procaryotic or eucaryotic mRNA,genomic sequences from viral or procaryotic DNA or RNA, and evensynthetic DNA sequences. A transcription termination sequence may belocated 3′ to the coding sequence.

[0061] A “promoter” is a nucleotide sequence which initiates andregulates transcription of a polypeptide-encoding polynucleotide.Promoters can include inducible promoters (where expression of apolynueleotide sequence operably linked to the promoter is induced by ananalyte, cofactor, regulatory protein, etc.), repressible promoters(where expression of a polynucleotide sequence operably linked to thepromoter is repressed by an analyte, cofactor, regulatory protein,etc.), and constitutive promoters. It is intended that the term“promoter” or “control element” includes full-length promoter regionsand functional (e.g., controls transcription or translation) segments ofthese regions.

[0062] “Operably linked” refers to an arrangement of elements whereinthe components so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a nucleic acidsequence is capable of effecting the expression of that sequence whenthe proper enzymes are present. The promoter need not be contiguous withthe sequence, so long as it functions to direct the expression thereof.Thus, for example, intervening untranslated yet transcribed sequencescan be present between the promoter sequence and the nucleic acidsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

[0063] The term “nucleic acid immunization” is used herein to refer tothe introduction of a nucleic acid molecule encoding one or moreselected antigens into a host cell for the in vivo expression of theantigen or antigens. The nucleic acid molecule can be introduceddirectly into the recipient subject by transdermal particle delivery.The molecule alternatively can be introduced ex vivo into cells whichhave been removed from a subject. In this latter case, cells containingthe nucleic acid molecule of interest are re-introduced into the subjectsuch that an immune response can be mounted against the antigen encodedby the nucleic acid molecule. The nucleic acid molecules used in suchimmunization are generally referred to herein as “nucleic acidvaccines.”

[0064] The term “solids content” indicates the amount of solids whichare either dissolved or suspended in the solvent(s) used.

[0065] The term “subject” refers to any member of the subphylum cordataincluding, without limitation, humans and other primates includingnon-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs; birds, including domestic,wild and game birds such as chickens, turkeys and other gallinaceousbirds, ducks, geese, and the like. The term does not denote a particularage. Thus, both adult and newborn individuals are intended to becovered. The methods described herein are intended for use in any of theabove vertebrate species, since the immune systems of all of thesevertebrates operate similarly.

[0066] The term “transdermal delivery” includes both transdermal(“percutaneous”) and transmucosal routes of administration, i.e.delivery by passage through the skin or mucosal tissue. See, e.g.,Transdermal Drug Delivery: Developmental Issues and ResearchInitiatives, Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989);Controlled Drug Delivery. Fundamentals and Applications, Robinson andLee (eds.), Marcel Dekker Inc., (1987); and Transdermal Delivery ofDrugs, Vols. 1-3, Kydonieus and Berner (eds.), CRC. Press, (1987).

[0067] B. General Methods

[0068] The invention is concerned with processes for producing powderssuitable for transdermal delivery via needleless syringe. As such, theparticles which make up the powdered composition must have sufficientphysical strength to withstand sudden acceleration to several times thespeed of sound and the impact with, and passage through, the skin andtissue.

[0069] In order to achieve powders having these properties, the processof the present invention is carried out using a solution or suspensioncontaining the desired pharmaceutical agent and having a total solidscontent of 20% by weight or more in the solvent system. Preferably thesolids content in the solvent system is 25% by weight or more, morepreferably 28% or 30% by weight or more and particularly preferably 40%by weight or more. The use of a solution or suspension having a solidscontent of 20% by weight or more minimises pore formation during thedrying process and increases the density of the particles formed. Thisincreased density is important in ensuring that the particles aresufficiently robust to survive the harsh conditions of transdermaldelivery via needleless syringe. The solids content of the solution ordispersion may be up to 50% by weight, up to 60% by weight or even up to70% by weight. The upper limit depends upon, for example, the particularcomponents of the solution or dispersion and the desired characteristicsof the resulting spray freeze-dried particles.

[0070] The pharmaceutical agent used to produce the powders of theinvention may be any small molecule drug substance, organic or inorganicchemical, vaccine, or peptide (polypeptide and/or protein) of interest.In particular embodiments, the pharmaceutical agent is abiopharmaceutical preparation of a peptide, polypeptide, protein or anyother such biological molecule. Exemplary peptide and proteinformulations include, without limitation, insulin; calcitonin;octreotide; endorphin; liprecin; pituitary hormones (e.g., human growthhormone and recombinant human growth hormone (hGH and rhGH), HMG,desmopressin acetate, etc); follicle luteoids; growth factors (such asgrowth hormone releasing factor (GHRF), somatostatin, somatotropin andplatelet-derived growth factor); asparaginase; chorionic gonadotropin;corticotropin (ACTH); erythropoietin (EPO); epoprostenol (plateletaggregation inhibitor); glucagon; interferons; interleukins; menotropins(urofollitropin, which contains follicle-stimulating hormone (FSH); andluteinizing hormone (LH)); oxytocin; streptokinase; tissue plasminogenactivator (TPA); urokinase; vasopressin; desmopressin; ACTH analogues;angiotensin II antagonists; antidiuretic hormone agonists; bradykininantagonists; CD4 molecules; antibody molecules and antibody fragments(e.g., Fab, Fab₂, Fv and sFv molecules); IGF-1; neurotrophic factors;colony stimulating factors; parathyroid hormone and agonists;parathyroid hormone antagonists; prostaglandin antagonists; protein C;protein S; renin inhibitors; thrombolytics; tumor necrosis factor (TNF);vaccines (particularly peptide vaccines including subunit and syntheticpeptide preparations); vasopressin antagonists analogues; and a-Iantitrypsin. Additionally, nucleic acid preparations, such as vectors orgene constructs for use in subsequent gene delivery, can be used.

[0071] Particularly suitable pharmaceutical agents for use herein areantigens. Any suitable antigen as defined herein may be employed. Theantigen may be a viral antigen. The antigen may therefore be derivedfrom members of the families Picornaviridae (e.g. polioviruses, etc.);Caliciviridae; Togaviridae (e.g. rubella virus, dengue virus, etc.);Flaviviridae; Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae(e.g. rabies virus, etc.); Filoviridae; Paramyxoviridae (e.g. mumpsvirus, measles virus, respiratory syncytial virus, etc.);Orthomyxoviridae (e.g. influenza virus types A, B and C., etc.);Bunyaviridae; Arenaviridae; Retroviradae (e.g. HTLV-I; HTLV-II; HIV-1and HIV-2); and simian immunodeficiency virus (SIV) among others.

[0072] Alternatively, viral antigens may be derived from apapillomavirus (e.g. HPV); a herpesvirus; a hepatitis virus, e.g.hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C. (HCV),the delta hepatitis virus (HDV), hepatitis E virus (HEV) or hepatitis Gvirus (HGV); and the tick-borne encephalitis viruses. See, e.g.Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2ndEdition (B. N. Fields and D. M. Knipe, eds. 1991) for a description ofthese viruses.

[0073] Bacterial antigens for use in the invention can be derived fromorganisms that cause diphtheria, cholera, tuberculosis, tetanus,pertussis, meningitis and other pathogenic states, includingMeningococcus A, B and C., Hemophilus influenza type B (HIB) andHelicobacter pylori. A combination of bacterial antigens may beprovided, for example diphtheria, pertussis and tetanus antigens.Suitable pertussis antigens are pertussis toxin and/or filamentoushaemagglutinin and/or pertactin, alternatively termed P69. Ananti-parasitic antigen may be derived from organisms causing malaria andLyme disease.

[0074] Antigens for use in the present invention can be produced using avariety of methods known to those of skill in the art. In particular,the antigens can be isolated directly from native sources, usingstandard purification techniques. Alternatively, whole killed,attenuated or inactivated bacteria, viruses, parasites or other microbesmay be employed. Yet further, antigens can be produced recombinantlyusing known techniques. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Vols. I and II (D. N. Glover et.1985).

[0075] Antigens for use herein may also be synthesised, based ondescribed amino acid sequences, via chemical polymer syntheses such assolid phase peptide synthesis. Such methods are known to those of skillin the art. See, e.g. J. M. Stewart and J. D. Young, Solid Phase PeptideSynthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill. (1984) and G.Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology,editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York,(1980), pp. 3-254, for solid phase peptide synthesis techniques; and M.Bodansky, Principles of Peptide Synthesis, Springer-Verlag Berlin (1984)and E. Gross and J. Meienhofer, Eds., The Peptides. Analysis, Synthesis,Biology, supra, Vol. 1, for classical solution synthesis.

[0076] The pharmaceutical agent may alternatively be a nucleic acidmolecule. The pharmaceutical agent can thus be a polynucleotide whichexpresses an antigen, such as in DNA immunization applications. Anexpression vector can thus be employed in which a nucleic acid sequenceencoding a desired polypeptide such as an antigen is operably linked toa promoter.

[0077] Typically, the nucleic acid molecule comprises a therapeuticallyrelevant nucleotide sequence for delivery to a subject. Thus, thenucleic acid molecule may comprise one or more genes encoding a proteindefective or missing from a target cell genome or one or more genes thatencode a non-native protein having a desired biological or therapeuticeffect (e.g., an antiviral function). The molecule may comprise asequence capable of providing immunity, for example an immunogenicsequence that serves to elicit a humoral and/or cellular response in asubject, or a sequence that corresponds to a molecule having anantisense or ribozyme function. For the treatment of genetic disorders,functional genes corresponding to genes known to be deficient in theparticular disorder can be administered to a subject.

[0078] Suitable nucleic acids for delivery include those used for thetreatment of inflammatory diseases, autoimmune, chronic and infectiousdiseases, including such disorders as AIDS, cancer, neurologicaldiseases, cardivascular disease, hypercholestemia; various blooddisorders including various anemias, thalassemia and hemophilia; geneticdefects such as cystic fibrosis, Gaucher's Disease, adenosine deaminase(ADA) deficiency, emphysema, etc. A number of antisense oligonucleotides(e.g., short oligonucleotides complementary to sequences around thetranslational initiation site (AUG codon) of an mRNA) that are useful inanitsense therapy for cancer and for viral diseases have been describedin the art. See, e.g., Han et al (1991) Proc. Natl. Acad. Sci USA88:4313; Uhlmann et al (1990) Chem. Rev. 90:543, Helene et al (1990)Biochim. Biophys. Acta. 1049:99; Agarwal et al (1988) Proc. Natl. Acad.Sci. USA 85:7079; and Heikkila et al (1987) Nature 328:445. A number ofribozymes suitable for use herein have also been described . See, e.g.,Chec et al (1992) J. Biol. Chem. 267: 17479 and U.S. Pat. No. 5,225,347to Goldberg et al.

[0079] For example, in methods for the treatment of solid tumors, genesencoding toxic peptides (i.e., chemotherapeutic agents such as ricin,diptheria toxin and cobra venom factor), tumor suppressor genes such asp53, genes coding for mRNA sequences which are antisense to transformingoncogenes, antineoplastic peptides such as tumor necrosis factor (TNF)and other cytokines, or transdominant negative mutants of transformingoncogenes, can be delivered for expression at or near the tumor site.

[0080] Similarly, nucleic acids coding for peptides known to displayantiviral and/or antibacterial activity, or stimulate the host's immunesystem, can also be administered. The nucleic acid may encode one of thevarious cytokines (or functional fragments thereof), such as theinterleukins, interferons, chemokines, chemotaxic factors, and colonystimulating factors. The nucleic acid may encode an antigen for thetreatment or prevention of a number of conditions including but notlimited to cancer, allergies, toxicity and infection by a pathogen suchas, but not limited to, fungus, viruses including Human Papiloma Viruses(HPV), HIV, HSV2/HSV1, influenza virus (types A, B and C), Polio virus,RSV virus, Rhinoviruses, Rotaviruses, Hepaptitis A virus, Norwalk VirusGroup, Enteroviruses, Astroviruses, Measles virus, Par Influenza virus,Mumps virus, Varicella-Zoster virus, Cytomegalovirus, Epstein-Barrvirus, Adenoviruses, Rubella virus, Human T-cell Lymphoma type I virus(HTLV-I), Hepatitis B virus (HBV), Hepatitis C. virus (HCV), Hepatitis Dvirus, Pox virus, Marburg and Ebola; bacteria including M. tuberculosis,Chlamydia, N. gonorrhoeae, Shigella, Salmonella, Vibrio Cholera,Treponema pallidua, Pseudomonas, Bordetella pertussis, Brucella,Franciscella tulorensis, Helicobacter pylori, Leptospria interrogaus,Legionella pnumophila, Yersinia pestis, Streptococcus (types A and B),Pneumococcus, Meningococcus, Hemophilus influenza (type b), Toxoplamagondic, Complybacteriosis, Moraxella catarrhalis, Donovanosis, andActinomycosis; fungal pathogens including Candidiasis and Aspergillosis;parasitic pathogens including Taenia, Flukes, Roundworms, Amebas,Giardia species, Cryptosporidium, Schitosoma species, Pneumocystiscarinii, Trichuriasis species, and Trichinella species. The nucleic acidmy also be used to provide a suitable immune response against numerousveterinary diseases, such as Foot and Mouth diseases, Coronavirus,Pasteurella multocida, Helicobacter, Strongylus vulgaris, Actinobacilluspleuropneumonia, Bovine viral diarrhea virus (BVDV), Klebsiellapneumoniae, E. Coli, Bordetella pertussis, Bordetella parapertussis andbrochiseptica. Thus in one aspect, the particles of the presentinvention may find use as a vaccine.

[0081] The invention will also find use in antisense therapy, e.g., forthe delivery of oligonucleotides able to hybridize to specificcomplementary sequences thereby inhibiting the transcription and/ortranslation of these sequences. Thus DNA or RNA coding for proteinsnecessary for the progress of a particular disease can be targeted,thereby disrupting the disease process. Antisense therapy, and numerousoligonucleotides which are capable of binding specifically andpredictably to certain nucleic acid target sequences in order to inhibitor modulate the expression of disease-causing genes are known andreadily available to the skilled practitioner. Uhlmann et al (1990) ChemRev. 90: 543, Neckers et al (1992) Int. Rev. Oncogenesis 3, 175; Simonset al (1992) Nature 359, 67; Bayever et al (1992) Antisense Res. Dev. 2:109; Whitesell et al (1991) Antisense Res. Dev. 1: 343; Cook et al(1991) Anti-cancer Drug Design 6: 585; Eguchi et al (1991) Ann. Rev.Biochem. 60: 631. Accordingly, antisense oligonucleotides capable ofselectively binding to target sequences in host cells are providedherein for use in antisense therapeutics.

[0082] The antigen or other pharmaceutical agent employed in the presentinvention may optionally be adsorbed into an adjuvant, such as analuminum salt adjuvant or calcium salt adjuvant. Alternatively, theantigen or other pharmaceutical agent may be used without an adjuvant.Suitable adjuvants include aluminium hydroxide, aluminum phosphate,aluminum sulfate and calcium phosphate.

[0083] The pharmaceutical agent is typically prepared as an aqueouspharmaceutical composition using a suitable aqueous carrier, along withsuitable excipients, protectants, solvents, salts, surfactants,buffering agents and the like. Suitable excipients can include anymaterials that are innocuous when administered to an individual, and donot significantly interact with the pharmaceutical agent in a mannerthat alters its pharmaceutical activity. Examples of normally employedexcipients include, but are not limited to, pharmaceutical grades ofdextrose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol,dextran, starch, cellulose, sodium or calcium phosphates, calciumcarbonate, calcium sulfate, sodium citrate, citric acid, tartaric acid,glycine, high molecular weight polyethylene glycols (PEG), andcombinations thereof. Suitable solvents include, but are not limited to,methylene chloride, acetone, methanol, ethanol, isopropanol and water.Typically, water is used as the solvent. Generally pharmaceuticallyacceptable salts having molarities ranging from about 1 mM to 2M can beused. Pharmaceutically acceptable salts include, for example, mineralacid salts such as hydrochlorides, hydrobromides, phosphates, sulfates,and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like. A thorough discussionof pharmaceutically acceptable excipients, vehicles and auxiliarysubstances is available in REMINGTON'S PHARMACEUTICAL SCIENCES (MackPub. Co., N.J. 1991), incorporated herein by reference.

[0084] The excipients chosen for use in the present invention may servespecific functions such as protein stabilization or surface protectionor may be used as bulking agents or to maintain low hygroscopicity ofthe powders. Although the invention is not limited to the use of anyspecific excipients, particularly suitable excipients includesaccharides, which may be amorphous or crystalline saccharides, polymersor amino acids or physiologically acceptable salts thereof. The use ofthese particular excipient compositions allow the particles to collapseand densify during freezing and therefore provide powders which areparticularly suitable for injection via a needleless syringe. Thesaccharide may be a monosaccharide, disaccharide or higher oligo- orpoly-saccharide. The excipients may be selected from carbohydrates,sugars and sugar alcohols. Such excipients may be crystalline oramorphous.

[0085] Preferably, one, two or three of these additives are present inthe solution or suspension in amounts of at least 15% by weight,preferably at least 20% by weight, such as at least 25%, 28% or 30% byweight and more preferably at least 40% by weight. Such additives may bepresent in amounts of up to 50% by weight, up to 60% by weight or evenup to 70% by weight. The upper limit depends upon, for example, theparticular additives used and the desired characteristics of theresulting spray freeze-dried particles. Most preferably one or twodifferent excipients are used.

[0086] Suitable amorphous saccharides include sugars. The amorphousexcipient may thus be selected from dextrose, sucrose, lactose,trehalose, cellobiose, raffinose, isomaltose and other carbohydratessuch as cyclodextrins. Such sugars are capable of stabilizing proteinsused as pharmaceutical agents during the spray-freeze-drying process andduring long-term storage.

[0087] Suitable crystalline carbohydrates, sugars and sugar alcoholsinclude mannitol, sorbitol and allitol. The combination of such acrystalline excipient with an amorphous excipient, typically anamorphous sugar, encourages the collapse of particles duringfreeze-drying and aids the formation of dense particles.

[0088] Suitable polymers include polysaccharides such as dextran ormaltodextran; starch; cellulose; polyvinylpyrrolidone (PVP),particularly PVP K-17; polyvinyl alcohol (PVA); and polyethylene glycols(PEG), particularly PEG with a molecular weight of 8,000. Dextrans arepreferred. Various grades of dextran are available. A suitable dextranmay have a molecular weight of greater than about 15,000 such as fromabout 15,000 to about 45,000, from about 60,000 to about 90,000 or fromabout 100,000 to about 200,000. The addition of a polymer as anexcipient tends to provide powders with improved flowability and mayalso provide increased protein stability.

[0089] Suitable amino acids and physiologically acceptable salts ofamino acids include glycine, alanine, glutamine, arginine, lysine andhistidine and salts thereof such as alkali or alkaline earth metalssalts such as sodium, potassium or magnesium salts or salts with otheramino acids such as glutamate or aspartate salts.

[0090] The most preferred combinations of excipients for use in thepresent invention include an amorphous saccharide with a crystallinesaccharide and optionally also a polymer and/or an amino acid or a saltthereof. The excipients may comprise an amorphous saccharide which istypically present in an amount of from 10 to 90% by weight, preferablyfrom 50 to 80% and more preferably from 60 to 75% by weight; and acrystalline saccharide which is typically present in an amount of from10 to 90% by weight, preferably from 20 to 50% and more preferably from25 to 40% by weight. This combination of excipients is preferably usedtogether with a surfactant which is typically present in an amount offrom 1 to 5% by weight. Alternatively, the additives may comprise anamorphous saccharide which is typically present in an amount of from 10to 80% by weight, preferably from 20 to 50%, more preferably from 25 to35% by weight; a crystalline saccharide which is typically present in anamount of from 10 to 80% by weight, preferably from 20 to 50%, morepreferably from 25 to 35% by weight; and a polymer or an amino acid orsalt thereof, each of which is present in an amount of from 10 to 80% byweight, preferably from 30 to 60%, more preferably from 30 to 50% byweight.

[0091] The most preferred additive combinations includetrehalose/mannitol, typically at a weight ratio of about 70/30;trehalose/mannitol/dextran, typically at a weight ratio of about30/30/40; trehalose/mannito/PVP at a weight ratio of about 30/30/40;trehalose/mannitol/PEG, typically at a weight ratio of about 30/30/40;or trehalose/mannitol/arginine glutamate, typically at a weight ratio ofabout 30/30/40. Particularly suitable particles can be prepared from anaqueous solution or dispersion of a pharmaceutical agent which furthercomprises trehalose, mannitol and dextran in a weight ratio of fromabout 3:3.:4 to about 4:4:3.

[0092] The use of these preferred additive combinations in the amountsdescribed above helps to provide particles with a high density. Thus,two major factors of the present invention act towards increasing thedensity of the particles. The first is the presence of at least 20% byweight of solids in the solution or suspension prior to freeze-dryingand the second is the selection of particular excipient compositions.

[0093] Whilst the excipient combinations described above are notessential for use in the present invention, they are particularlypreferred when the total amount of solids in the solution or suspensionis close to 20% by weight, such as less than 40% by weight, inparticular less than 30% or less than 25% by weight. When the solutionor suspension has a solids content as low as this, the density of theparticles, whilst sufficient for the purposes of the invention, candesirably be increased further by use of the above-described excipientcompositions. However, if the solids content is above 30% by weight ormore preferably above 40% by weight, the particles produced will besufficiently dense, so that the extra density obtained by using thepreferred excipients in the ratios described above is less important.

[0094] The particles of the invention may additionally contain otheradditives such as surfactants. Suitable surfactants for use in thepresent invention include non-ionic surfactants. The surfactant may be apolysorbate such as Tween 20 and Tween 80, a Pluronics surfactant suchas F68 or a Span surfactant. The surfactant can be used in combinationwith any of the above-named combinations of excipients.

[0095] The particles of the invention are formed by first dissolving orsuspending the pharmaceutical agent, and any required additives, inwater. The aqueous solution or suspension formed must have a totalsolids content in the water of at least 20% by weight. The aqueoussolution or suspension is then spray freeze-dried. Any known techniquein the art (for example the methods described by Mumenthaler et al, Int.J. Pharmaceutics (1991) 72, pages 97-110 and Maa et al, Phar. Res.(1999) Vol. 16, page 249) may be used to carry out the sprayfreeze-drying step.

[0096] A typical spray freeze-drying technique involves atomising theaqueous solution or suspension into a liquified gas, which is generallyunder stirring. The liquified gas can be liquid argon, liquid nitrogen,or any other gas that results in the immediate freezing of the atomiseddroplets of the aqueous solution or suspension. Preferably the liquifiedgas is an inert liquified gas such as liquid nitrogen.

[0097] The liquified gas containing the frozen droplets of the aqueoussolution or suspension is then freeze-dried. It is not contacted with anorganic solvent such as methanol, ethanol, ethyl ether, acetone,pentane, methylene chloride, chloroform or ethyl acetate. Drying is nottherefore conducted according to the procedures described in U.S. Pat.No. 5,019,400.

[0098] Typically, the liquified gas containing the frozen droplets istransferred into a lyophiliser for freeze-drying. The liquified gascontaining the frozen droplets is usually poured into a metal tray andintroduced into the lyophiliser. The frozen droplets are freeze-dried inthe tray. The liquid nitrogen evaporates and the frozen water containedin the droplets is removed by sublimation. The resulting particles arecollected. They can be washed as desired.

[0099] In more detail, the liquified gas containing frozen droplets ofthe atomized solution or suspension is held at reduced temperature, forexample from about −60° C. to −40° C. Typically, that is followed bytwo-stage vacuum drying preferably under a pressure of from about 20 to500 mT (2.666 to 66.65 Pa). The first drying stage is normally performedat a reduced temperature such as from about −50° C. to 0C., for a periodof about 4 to 24 hours. Frozen water is removed by ice sublimation. Inthe second drying stage, drying is normally performed at a highertemperature such as from about 5 to 30°C. at a lower pressure,preferably less than 100 mT down to about 10 mT, for a period of about 5to 24 hours. The precise spray freeze-drying conditions used may beselected according to the desired properties of the particles to beproduced. Thus, the temperatures, pressures and other conditions may bevaried as desired.

[0100] Preferably, the nozzle used to atomise the solution or suspensionis an ultrasonic nozzle. This has the advantage of being a mild processwhich generates little stress to the biomolecules which are frequentlyused as therapeutic agents in the present invention. In addition, use ofan ultrasonic nozzle eliminates the need for pressurized gas to assistthe liquid feed which, in turn helps increase the yield of the process.The predominant variable for control of droplet size in an ultrasonicnozzle system is the nozzle frequency, although surface tension,viscosity and density of the liquid feed are additional variables thatcan be manipulated to control droplet size. Thus, for example, smallerparticles may be produced by increasing the nozzle frequency and viceversa. When using the ultrasonic nozzle system. am accurate. low-pulsefeed pump can be used to delivery the liquid feed, wherein such pumpsare particularly well suited when operating at low feed rates (e.g.,about 3 to 5 ml per minute) normally associated with laboratory-scaleparticle production. It has been found that atomization proceeds well atabout 1 to 2 Watts above the “critical power” level of the low-pulsepump system. In some drying cycles, it has been found that operation atabout 2.9 to 3.1 Watts allows for the most efficient atomization,however the exact operating conditions will also depend upon the liquidcharacteristics of the feed (viscosity, density, total solids content,surface tension, etc.).

[0101] A dual spray freeze-drying process may also be used. This processis particularly useful when the pharmaceutical agent is a protein havinga low water solubility. This dual process comprises spray freeze-dryingthe liquid protein to form a dry powder. This powder is thenreconstituted in water to provide a suspension having the desired solidcontent and spray freeze-dried for a second time.

[0102] The spray freeze-dried particular that are obtained according tothe invention can be collected, washed and dried. The dried particlescan then be sieved to obtained particles of the desired size.

[0103] The particles of the invention have a size appropriate forhigh-velocity transdermal delivery to a subject, typically across thestratum corneum or a transmucosal membrane. The mass mean aerodynamicdiameter (MMAD) of the particles is from about 0.1 to 250 μm. The MMADmay be from 5 to 100 μm or from 10 to 100 μm, preferably from 10 to 70μm or from 20 to 70 μm. Generally, less than 10% by weight of theparticles have a diameter which is at least 5 μm more than the MMAD orat least 5 μm less than the MMAD. Preferably, no more than 5% by weightof the particles have a diameter which is greater than the MMAD by 5 μmor more. Also preferably, no more than 5% by weight of the particleshave a diameter which is smaller than the MMAD by 5 μm or more. Theparticle size is controllable by varying the frequency of the ultrasonicnozzle used to atomise the solution or suspension.

[0104] The particles typically have an envelope density of from 0.5 to25 g/cm³, preferably from 0.6 to 1.8 g/cm³. More preferably the envelopedensity is from 0.7 to 1.5 g/cm³. The attainment of the above minimumenvelope density value is particularly preferred, since particles with alower density tend to perform poorly during penetration of the skin andmay not be suitable for use in a transdermal needleless injectionsystem. The particles have a low porosity, wherein typically at least70%, at least 80%, at least 85% or at least 90% of the particle is noroccupied by pores.

[0105] While the shape of the individual particles may vary when viewedunder a microscope, the particles are preferably substantiallyspherical. The average ratio of the major axis:minor axis is typicallyfrom 3:1 to 1:1, for example from 2:1 to 1:1.

[0106] The individual particles of the powder have a substantiallyspherical aerodynamic shape with a substantially uniform, nonporoussurface. The particles will also have a particle penetration energysuitable for transdermal delivery from a needleless syringe device. Theparticles should also be free-flowing under a dry environment. Forexample, the particles should flow freely in a vial upon rotation at arelative humidity of less than 30%. Preferably, the particles arefree-flowing under ambient conditions, such as a relative humidity ofless than 60%. The moisture content of the particles should preferablybe less than 5%, more preferably less than 2%, after freeze-drying, andthis level of moisture should be maintained during storage at less than30% humidity for, for example, at least one month and preferably muchlonger.

[0107] A detailed description of needleless syringe devices useful inthis invention is found in the prior art, as discussed herein. Thesedevices are referred to as needleless syringe devices and representativeof these devices are the dermal PowderJect® needleless syringe deviceand the oral PowderJect® needleless syringe device (PowderJectTechnologies Limited, Oxford, UK). By using these devices, an effectiveamount of the powder of the invention is delivered to the subject. Aneffective amount is that amount needed to deliver a sufficient quantityof the desired antigen to achieve vaccination. This amount will varywith the nature of the antigen and can be readily determined throughclinical testing based on known activities of the antigen beingdelivered. The “Physicians Desk Reference” and “Goodman and Gilman's ThePhamacological Basis of Therapeutics” are useful for the purpose ofdetermining the amount needed.

[0108] Needleless syringe devices for delivering particles were firstdescribed in commonly owned U.S. Pat. No. 5,630,796 to Bellhouse et al,incorporated herein by reference. Although a number of specific deviceconfigurations are now available, such devices are typically provided asa pen-shaped instrument containing, in linear order moving from top tobottom, a gas cylinder, a particle cassette or package, and a supersonicnozzle with an associated silencer element. An appropriate powder (inthe present case, a spray freeze-dried powder of the invention) isprovided within a suitable container, e.g., a cassette formed by tworupturable polymer membranes that are heat-sealed to a washer-shapedspacer to form a self-contained sealed unit. Membrane materials can beselected to achieve a specific mode of opening and burst pressure thatdictate the conditions at which the supersonic flow is initiated. Inoperation, the device is actuated to release the compressed gas from thecylinder into an expansion chamber within the device. The released gascontacts the particle cassette and, when sufficient pressure is builtup, suddenly breaches the cassette membranes sweeping the particles intothe supersonic nozzle for subsequent delivery. The nozzle is designed toachieve a specific gas velocity and flow pattern to deliver a quantityof particles to a target surface of predefined area. The silencer isused to attenuate the noise produced by the transient supersonic flowand/or membrane rupture.

[0109] A second needleless syringe device for delivering particles isdescribed in commonly owned International Publication No. WO 96/20022.This delivery system also uses the energy of a compressed gas source toaccelerate and deliver powdered compositions; however, it isdistinguished from the system of US Patent No. 5,630,796 in its use of ashock wave instead of gas flow to accelerate the particles. Moreparticularly, an instantaneous pressure rise provided by a shock wavegenerated behind a flexible dome strikes the back of the dome, causing asudden eversion of the flexible dome in the direction of a targetsurface. This sudden eversion catapults a powdered composition (which islocated on the outside of the dome) at a sufficient velocity, thusmomentum, to penetrate target tissue, e.g., oral mucosal tissue. Thepowdered composition is released at the point of full dome eversion. Thedome also serves to completely contain the high-pressure gas flow, whichtherefore does not come into contact with the tissue. Because the gas isnot released during this delivery operation, the system is inherentlyquiet. This design can be used in other enclosed or otherwise sensitiveapplications for example, to deliver particles to sites reached byminimally invasive surgery.

[0110] In yet a further aspect of the invention, single unit dosages ormultidose containers, in which the powder of the invention may bepackaged prior to use, can comprise a hermetically sealed containerenclosing a suitable amount of the powder that makes up a suitable dose.The powder can be packaged as a sterile formulation, and thehermetically sealed container can thus be designed to preserve sterilityof the formulation until use. If desired, the containers can be adaptedfor direct use in the above-referenced needleless syringe systems.

[0111] Powders of the present invention can thus be packaged inindividual unit dosages for delivery via a needleless syringe. As usedherein, a “unit dosage” intends a dosage receptacle containing atherapeutically effective amount of a powder of the invention. Thedosage receptacle typically fits within a needleless syringe device toallow for transdermal delivery from the device. Such receptacles can becapsules, foil pouches, sachets, cassettes or the like.

[0112] The container in which the powder is packaged can further belabeled to identify the composition and provide relevant dosageinformation. In addition, the container can be labeled with a notice inthe form prescribed by a governmental agency, for example the U.S. Foodand Drug Administration, wherein the notice indicates approval by theagency under U.S. Federal Law of the manufacture, use or sale of thepowder contained therein for human administration.

[0113] The actual distance which the delivered particles will penetratea target surface depends upon particle size (e.g., the nominal particlediameter assuming a roughly spherical particle geometry), particledensity, the initial velocity at which the particle impacts the surface,and the density and kinematic viscosity of the targeted skin tissue. Inthis regard, optimal particle densities for use in needleless injectiongenerally range between about 0.5 and 25 g/cm³, preferably between about0.7 and 1.5 g/cm³, and injection velocities generally range betweenabout 100 and 3,000 m/sec. With appropriate gas pressure, particleshaving an average diameter of 10-70 μm can be accelerated through thenozzle at velocities approaching the supersonic speeds of a driving gasflow.

[0114] If desired, the needleless syringe systems can be provided in apreloaded condition containing a suitable dosage of the powder of theinvention. The loaded syringe can be packaged in a hermetically sealedcontainer, which may further be labeled as described above.

[0115] A number of novel test methods have been developed, orestablished test methods modified, in order to characterize performanceof a needleless syringe device. These tests range from characterizationof the powdered composition, assessment of the gas flow and particleacceleration, impact on artificial or biological targets, and measuresof complete system performance. One, several or all of the followingtests can thus be employed to assess the physical and functionalsuitability of the powder of the invention for use in a needlelesssyringe system.

[0116] Assessment of Effect on Artificial Film Targets

[0117] A functional test that measures many aspects of powder injectionsystems simultaneously has been designated as the “metallized film” or“penetration energy” (PE) test. It is based upon the quantitativeassessment of the damage that particles can do to a precision thin metallayer supported by a plastic film substrate. Damage correlates to thekinetic energy and certain other characteristics of the particles. Thehigher the response from the test (i.e., the higher the filmdamage/disruption) the more energy the device has imparted to theparticles. Either electrical resistance change measurement or imagingdensitometry, in reflectance or transmission mode, provide a reliablemethod to assess device or formulation performance in a controllable andreproducible test.

[0118] The film test-bed has been shown to be sensitive to particledelivery variations of all major device parameters including pressure,dose, particle size distribution and material, etc. and to beinsensitive to the gas. Aluminum of about 350 Angstrom thickness on a125 μm polyester support is currently used to test devices operated atup to 60 bar helium pressure.

[0119] Assessment of Impact Effect on Engineering Foam Targets

[0120] Another means of assessing particle performance when deliveredvia a needleless syringe device is to gauge the effect of impact on arigid polymethylimide foam (Rohacell 5 IIG, density 52 kg/m³, Rohm TechInc., Malden, Mass.). The experimental set-up for this test is similarto that used in the metallized film test. The depth of penetration ismeasured using precision calipers. For each experiment a processedmannitol standard is run as comparison and all other parameters such asdevice pressure, particle size range, etc., are held constant. Data alsoshow this method to be sensitive to differences in particle size andpressure. Processed mannitol standard as an excipient for drugs has beenproven to deliver systemic concentrations in preclinical experiments, sothe relative performance measure in the foam penetration test has apractical in vivo foundation. Promising powders can be expected to showequivalent or better penetration to mannitol for anticipation ofadequate performance in preclinical or clinical studies. This simple,rapid test has value as a relative method of evaluation of powders andis not intended to be considered in isolation.

[0121] Particle Attrition Test

[0122] A further indicator of particle performance is to test theability of various candidate compositions to withstand the forcesassociated with high-velocity particle injection techniques, that is,the forces from contacting particles at rest with a sudden, highvelocity gas flow, the forces resulting from particle-to-particle impactas the powder travels through the needleless syringe, and the forcesresulting from particle-to-device collisions also as the powder travelsthrough the device. Accordingly, a simple particle attrition test hasbeen devised which measures the change in particle size distributionbetween the initial composition, and the composition after having beendelivered from a needleless syringe device.

[0123] The test is conducted by loading a particle composition into aneedleless syringe as described above, and then discharging the deviceinto a flask containing a carrier fluid in which the particularcomposition is not soluble (e.g., mineral oil, silicone oil, etc.). Thecarrier fluid is then collected, and particle size distribution in boththe initial composition and the discharged composition is calculatedusing a suitable particle sizing apparatus, e.g., an AccuSizer® model780 Optical Particle Sizer. Compositions that demonstrate less thanabout 50%, more preferably less than about 20% reduction in mass meandiameter (as determined by the AccuSizer apparatus) after deviceactuation are deemed suitable for use in the needleless syringe systemsdescribed herein.

[0124] Delivery to Human Skin in vitro and Transepidermal Water Loss

[0125] For a powder performance test that more closely parallelseventual practical use, candidate powder compositions can be injectedinto dermatomed, full thickness human abdomen skin samples. Replicateskin samples after injection can be placed on modified Franz diffusioncells containing 32° C. water, physiologic saline or buffer. Additivessuch as surfactants may be used to prevent binding to diffusion cellcomponents. Two kinds of measurements can be made to assess performanceof the formulation in the skin.

[0126] To measure physical effects, i.e. the effect of particleinjection on the barrier function of skin, the transepidermal water loss(TEWL) can be measured. Measurement is performed at equilibrium (about 1hour) using a Tewameter TM 210® (Courage & Khazaka, Koln, Germany)placed on the top of the diffusion cell cap that acts like a˜12 mmchimney. Larger particles and higher injection pressures generateproportionally higher TEWL values in vitro and this has been shown tocorrelate with results in vivo. Upon particle injection in vitro TEWLvalues increased from about 7 to about 27 (g/m²h) depending on particlesize and helium gas pressure. Helium injection without powder has noeffect. In vivo, the skin barrier properties return rapidly to normal asindicated by the TEWL returning to pretreatment values in about 1 hourfor most powder sizes. For the largest particles, 53-75 μm, skin samplesshow 50% recovery in an hour and full recovery by 24 hours.

[0127] Delivery to Human Skin in vitro and Drug Diffusion Rate

[0128] To measure the formulation performance in vitro, the drug orantigen component(s) of candidate powders can be collected by completeor aliquot replacement of the Franz cell receiver solution atpredetermined time intervals for chemical assay using HPLC. or othersuitable analytical technique. Concentration data can be used togenerate a delivery profile and calculate a steady state permeationrate. This technique can be used to screen formulations for earlyindication of drug or antigen binding to skin, drug or antigendissolution, efficiency of particle penetration of stratum corneum,etc., prior to in vivo studies.

[0129] These and other qualitative and quantitative tests can be used toassess the physical and functional suitability of the present powdersfor use in a high-velocity particle injection device. It is preferred,though not required, that the particles of a powder have the followingcharacteristics: a substantially spherical shape (e.g. an aspect ratioas close as possible to 1); a smooth surface; a suitable active loadingcontent; less than 20% reduction in particle size using the particleattrition test; an envelope density as close as possible to the truedensity of the constituents (e.g. greater than about 0.5 g/ml); and aMMAD of about 20 to 70 μm with a narrow particle size distribution. Thecompositions are typically free-flowing (e.g. free-flowing after 8 hoursstorage at 50% relative humidity and after 24 hours storage at 40%relative humidity). All of these criteria can be assessed using theabove-described methods, and are further detailed in the followingpublications, incorporated herein by reference. Etzler et al (1995)Part. Part. Syst. Charact.12:217; Ghadiri, et al (1992) IFPRI FinalReport, FRR 16-03 University of Surrey, UK; Bellhouse et al (1997)“Needleless delivery of drugs in dry powder form, using shock waves andsupersonic gas flow,” Plenary Lecture 6, 21^(st) International Symposiumon Shock Waves, Australia; Kwon et al (1998) Pharm. Sci. suppl. 1 (1),103; and Burkoth et al. (1999) “Transdermal and Transmucosal PowderedDrug Delivery,” in Critical Reviews in the Therapeutic Drug CarrierSystems 16(4):331-384, STephen Bruck Ed., Begell House Inc., New York,N.Y.

[0130] A powder of the invention may alternatively be used to vaccinatea subject via other routes. For this purpose, the powder may be combinedwith a suitable carrier or diluent such as Water for Injections orphysiologically saline. The resulting vaccine composition is typicallyadministered by injection, for example subcutaneously orintramuscularly.

[0131] Whichever route of administration is selected, an effectiveamount of antigen is delivered to the subject being vaccinated.Generally from 50 ng to 1 mg and more preferably from 1 μg to about 50μg of antigen will be useful in generating an immune response. The exactamount necessary will vary depending on the age and general condition ofthe subject to be treated, the particular antigen or antigens selected,the site of administration and other factors. An appropriate effectiveamount can be readily determined by one of skill in the art.

[0132] Dosage treatment may be a single dose schedule or a multiple doseschedule. A multiple dose schedule is one in which a primary course ofvaccination may be with 1-10 separate doses, followed by other dosesgiven at subsequent time intervals, chosen to maintain and/or reinforcethe immune response, for example at 1-4 months for second dose and, ifneeded, a subsequent dose(s) after several months. The dosage regimenwill also, at least in part, be determined by the need of the subjectand be dependent on the judgement of the practitioner. Vaccination willof course generally be effected prior to primary infection with thepathogen against which protection is desired.

[0133] C. Experimental

[0134] Below are examples of specific embodiments for carrying out thepresent invention. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0135] Efforts have been made to ensure accuracy with respect to numbersused (e.g., amounts, temperatures, etc.), but some experimental errorand deviation should, of course, be allowed for.

EXAMPLE 1 Spray Freeze Drying (“SFD”) of Hepatitis B VaccineCompositions

[0136] 1.1 Objectives:

[0137] To assess the SFD method for preparing Hepatitis-B vaccinepowders and to particularly assess the following formulation parameters:(a) the concentration effect on vaccine; and (b) the solid contenteffect on particle density.

[0138] 1.2 Materials:

[0139] Hepatitis B surface antigen (HBsAg, Lot # 64850) was obtainedfrom Rhein Americana S.A. (Argentine Republic). Nominal concentration ofthe antigen was reported as 1.37 mg/mL in the attached Quality ControlCertificate of Analysis. The materials used in the study are reportedbelow in Table 1.1. TABLE 1.1 Materials used in the study Material Lot #Source Comment Hepatitis-B surface 64850 Rhein Americana Nominal antigenS.A. (Argentina concentration at Republic) 1.37 mg/mL (20 μg per humandose), (“HBsAg”) Alum-adjuvanted 17 Rhein Americana 20 ug HBsAg HBsAgS.A. (Argentina adsorbed to Republic) 0.44 mg of alum per vial (˜1.0mL), (“HBsAg/alum”) Dextran (MW 10 69H1273 Sigma kDa) (St. Louis, MO)Mannitol 127H0960 Sigma (St. Louis, MO) trehalose dihydrate 28H3797Sigma (St. Louis, MO)

[0140] 1.3 Formulations:

[0141] Table 1.2, below, shows the percent (w/w) composition of thestarting liquid formulations, and the targeted composition in 1.0-mg ofpayload. The key excipients were trehalose, mannitol, and dextran of 10kDa molecular weight. Trehalose was used since it is capable ofstabilizing the protein during lyophilization and long-term storage, andthe combination of mannitol and trehalose allows dense particles to beproduced during spray-freeze-drying. Dextran was used to improve thepowder's flowability, the mechanical properties of the particles, andpossibly the overall vaccine composition stability. All liquidformulations were targeted to have a total solid content of 30%. TABLE1.2 Composition (% w/w) of HBsAg and HBsAg/alum formulations Batch #138-20-1 138-20-2 138-20-3 138-20-4A 138-20-4B 138-16-1 138-20-5 Solidcontent (%) 30 30 30 30 5 30 10 Conc. Of HBsAg 403.5 407.5 403.5 403.5403.5 15.0 5.0 (human dose/mL) Batch size (dose) 500 500 500 400 300 400300 HBsAg solution 133.2 317.0 622.4 989.7 742.3 1891.0 5400.0 (mg)Trehalose 164.5 164.4 163.4 143.1 107.3 251.2 188.0 (dihydrate) Mannitol199.8 199.23 198.0 172.7 129.5 303.9 228.0 Dextran (10 kDa) 150.0 148.7148.5 129.1 96.9 228.9 172.2

[0142] 1.4 Methods:

[0143] 1.4.1 Vaccine Concentration

[0144] A centrifugal filter device, Centriprep, with a 10 kD limitregenerated cellulose membrane and a 15 mL sample container (Millipore,Bedford, Mass.) was used to concentrate the alum-free bulk vaccine. Thefilter was rinsed twice with 15 mL of nanopure water to remove the traceamount of glycerin present in the filter by centrifugation at 3000 rpmat 5-10° C. (Allegra 6®, Beckman, Fullerton, Calif.). The filter wasthen rinsed once with a blank buffer at the specified pII. The vaccinesolution was centrifuged until a desired amount of permeate wascollected. Three separate batches were prepared for three powderformulations.

[0145] The alum-adjuvanted bulk vaccine was pooled from vials andcentrifuged (Allegra 6R, Beckman, Palo Alto, Calif.) at 3,000 rpm for 10minutes. The cake was re-suspended with fresh water by vortex prior toformulating with excipients.

[0146] 1.4.2 Spray-Freeze-Drying

[0147] The SFD apparatus featured an ultrasonic atomizer (Sono-TekCorporation, Milton, N.Y.) having a spraying nozzle (Model #05793) and apower supply (Model #06-05108). The nozzle was equipped with aquasi-electric quartz crystal capable of vibrating at a specificfrequency that determines the size of the droplets. The frequency of 60kHz produces droplets mostly within the range of 20-80 μm. Circularmetal pans (16-cm in diameter by 6-cm in height) were used to containthe liquid nitrogen. For the lyophilization, a shelf freeze dryer (Model#TDS2C2B5200, Dura-Stop, FTS System, Stone Ridge, N.Y.) was used. Thisdryer can hold six metal pans in one batch run. Other apparatus includeda magnetic stirrer with magnetic stir bars, and a peristaltic pump(Model #77120-70, MasterFlex C/L, Barnant Company, Barrington, Ill.).

[0148] The liquid feed (vaccine formulation) was delivered by theperistaltic pump at a flow rate of 1.5 mL/min into the ultrasonicatomizer (60 kHz) where the liquid formulation was sprayed into theliquid N₂-containing pan. After spraying, the pan containing frozenparticles in liquid nitrogen was transferred to a pre-cooled freezedryer (−55° C.). Liquid nitrogen evaporated in a few minutes. Thefreeze-drying cycle was varied to control particle characteristics, buta typical lyophilization cycle is listed below in Table 1.3. TABLE 1.3Freeze-drying cycle Stage/ Cycle Conditions Freezing pre-cool shelftemperature (ST) = −50° C. ramp at 1.0° C./min to ST = −55° C., hold for15 min wait for product temp (PT) = −48° C., hold for 120 min Primarycondenser/vacuum (C/V) switched “on” Drying when condenser temp. reaches−40 C., vacuum pump turned on wait for chamber vacuum to reach 150 mTwait for foreline vacuum to reach 100 mT ramp at 1.0° C./min to ST =−10° C., hold for 24 hours Secondary ramp at 1.0° C./min to ST = 20° C.,hold for 24 hours Drying

[0149] After drying, the powder-containing pan was transferred into adry box purged with nitrogen with relative humidity initially held at<20%. The relative humidity was increased gradually to ˜40% toequilibrate the powder prior to powder collection.

[0150]1.5 Powder Characterization:

[0151] 1.5.1 Particle Size Analysis

[0152] The mean geometric diameter of the particles in the volumedistribution was determined using an AccuSizer 780 (Particle SizingSystems, Santa Barbara, Calif.). Based on the light obscurationtechnique, the AccuSizer determines the particle size distributionwithout assuming the shape distribution of the particle. In addition,the size of the particle population between 10% and 90% (volume) wasalso determined. Each analysis required approximately 5 mg of the powdersample. Powder samples were suspended in light mineral oil and sonicatedfor 5-10 seconds to remove agglomeration of particles before analysis.

[0153] The mean acrodynamic diameter of the particles in the volumedistribution was also determined using a dry powder dispersion-basedparticle size analyser (Aerosizer, API). Here again, each analysisrequired approximately 5 mg of the powder sample.

[0154] As reported below in Table 1.4, the particle size of all powderstested fell in the range of 20-60 μm based on Aerosizer measurement.TABLE 1.4 Particle size result by Aerosizer D50% (median) D10% D90%Batch # Formulation size (μm) (μm) (μm) 138-20-1 2 μg HBsAg 40.1 25.157.4 138-20-2 5 μg HBsAg 39.4 25.3 53.1 138-20-3 10 μg HBsAg 36.7 23.651.7 138-20-4A 20 μg HBsAg 36.9 24.2 50.5 138-20-4B 20 μg HBsAg 39.826.9 54.6 (FD/C/S) 138-16-1 2 μg HBsAg/ 41.0 26.2 55.9 50 μg alum138-20-5C 2 μg HBsAg/ 42.9 31.3 54.3 50 μg g alum SFD/C/S 138-16-1C 2 μgHBsAg/ na na na 50 μg alum SFD/sieved

[0155] 1.5.2 Image Analysis

[0156] Visual analysis of the particles was performed using an opticalmicroscope (Model DMR, Leica, Germany) with 10×-eyepiece lens and10×-objective lens. The system was equipped with a Polaroid camerasystem for image output. Digital images were captured and stored.

[0157] The image pictures of selected formulations are presented inFIGS. 1a-1 d. As can be seen, particle shape was generally spherical,and the estimated particle size consistent with that measured by theAerosizer.

[0158] 1.5.3 Scanning Electron Microscopy (SEM)

[0159] SEM was performed on an Amray 1810 T instrument after powdersamples were sputeer-coated with gold. Measurement was courtesy of Prof.Geoffrey Lee and Ms. Christine Sonner of the Friedrich-AlexanderUniversity in Erlangen, Germany.

[0160] The SEM micrographs are presented in FIGS. 2a-2 d. As can beseen, all SFD particles (FIGS. 2a and 2 b) were spherical in shape withwrinkled morphology, suggesting particle shrinkage during drying, whilethe particles that were formed by a compress, grind and sieve (C/G/S)technique had an irregular shape and surface morphology.

[0161] 1.5.4 Tap Density

[0162] Each powder sample was weighed in a glass vial and gently tappedagainst the lab bench for 20 times. By visual inspection, water of anequivalent volume to that of the powder was placed into an empty vial ofthe same type. The tap density of the powder sample could be calculatedby dividing the powder sample weight with the water sample weight(assuming water density=1 g/mL).

[0163] As summarized in Table 1.5 below, the tap density of all powderswas >0.4 g/mL. Higher tap densities are achievable by using a highersolid content, for example, tap densities of >0.6 g/mL were seen whenthe solid content was raised to 35%. TABLE 1.5 Tap density result Tapdensity Batch # Formulation (g/mL) 138-20-1 2 μg HBsAg 0.52 138-20-2 5μg HBsAg 0.51 138-20-3 10 μg HBsAg 0.43 138-20-4A 20 μg HBsAg 0.44138-20-4B 20 μg HBsAg 0.49 (FD and C/G/S) 138-16-1 2 μg HBsAg/ 0.51 50μg alum 138-20-5C 2 μg HBsAg/ 0.42 50 μg alum SFD and C/G/S 138-16-1C 2μg HBsAg/ 0.51 50 μg alum SFD/sieved

[0164] 1.5.5 Moisture Content Analysis

[0165] Approximately 4-5 mg of the powder sample was recovered from thetrilaminate cassettes and transferred into an aluminium weighing-vesseland the weight was recorded. The sample was loaded into a Karl FisherCoulometer (Model 737, Brinkmann) equipped with a drying oven (Model707). The sample was heated to 150°C. for 150 sec with a gas flow rateof 100 mL/min within the drying oven.

[0166] As reported below in Table 1.6, the moisture content in thepowders ranged from 3.3% to 4.1%. Under the same drying condition, therewas no clear correlation between moisture content and chemicalcomposition. TABLE 1.6 Moisture content result Moisture content Batch #Formulation (%) 138-20-1 2 μg HBsAg 3.9 138-20-2 5 μg HBsAg 3.6 138-20-310 μg HBsAg 4.1 138-20-4A 20 μg HBsAg 3.7 138-20-4B 20 μg HBsAg 3.3(FD/C/S) 138-16-1 2 μg HBsAg/ 3.9 50 μg alum 138-20-5C 2 μg HBsAg/ 3.350 μg alum SFD/C/S 138-16-1C 2 μg HBsAg/ 3.9 50 μg alum SFD/sieved

[0167] 1.5.6 Particle Attrition Testing

[0168] This method allows particle attrition arising from, e.g.,particle collisions within the powder injection device to be quanified.This method can be used to assess particle integrity upon contacting theskin prior to penetration indirectly through measuring the mean particlesize reduction and particle size distribution changes of the powderafter firing from a needleless syringe (powder injection) device. Thecontrol sample (prior to firing) was prepared by suspending 5 mg ofpowder in mineral oil (about 30 mL) in a 40 mL container. The mixturewas vortexed/sonicated to make a homogeneous suspension. The particlesize distribution was measured using a particle size analyzer (AccuSizer780, Particle Sizing Systems, Santa Barbara, Calif.). For thepost-firing sample, the powder was actuated from the device (PowderJect®powder injection device, PowderJect Pharmaceuticals plc, Oxford, UnitedKingdom), using trilaminate cassettes and 20 μm polycarbonate membranesto contain the powder, 5 shots at a payload of 2 mg each) into a 1 LErlenmeyer flask. The flask was coated with 25 mL of light mineral oiland the top was covered with a latex sheet with a {fraction (3/16)} inchhole in the center. The flask sat for about 2-3 minutes until no flyingparticles could be seen. The interior wall of the flask was washed with25 mL of fresh mineral oil and vortexed or shaken vigorously toestablish a homogeneous suspension. Fifteen mL of the powder suspensionwas further diluted with 15-mL of mineral oil and dispensed into a 40 cctube. Both samples before and after attrition were subject to lightobscuration analysis. The experiment was repeated three times and eachsample was measured in triplicate. The particle size distributionprofile from the post-firing sample was compared with that from thecontrol sample. The decrease in the ratio of the respective mean sizerepresents the extent of particle attrition.

[0169] As reported below in Table 1.7, particle size reduction for 3selected formulations was found to be similar and all less than 30%.TABLE 1.7 Particle attrition test results Mean Particle Size MeanParticle Size of pre- of post- % Reduction Samples Actuation (μm)Actuation (μm) in Mean Size 138-20-3 53.2 41.8 21.5 138-20-4A 53.9 41.423.2 1380-20-4B 55.3 40.2 27.3

[0170]1.5.7 Reconstitution of Alum-Containing Powder

[0171] The alum-containing powder sample (2-mg) was dissolved(reconstituted) in 0.5-mL of water. The optical image of the liquidsuspension was taken. This procedure allows alum coagulation to bedetermined.

[0172] Two alum-containing formulations (batch numbers 138-16-1 &138-20-5C) were reconstituted in water and their optical micrographs areshown in FIGS. 3a and 3 b. The sandy appearance of batch no. 138-16-1(FIG. 3a) suggests that alum coagulation after SFD is minimal. This isdue the fast-freezing phenomenon associated with the SFD process. Thereis slight coagulation seen with batch no. 138-20-5C. (FIG. 3b) that hasthe same formulation as batch 138-16-1 but has been further processedusing a compress, grind, and sieve technique. This is consistent with aprevious observation that compression caused alum to coagulate slightly.

[0173] 1.5.8 SDS-PAGE Analysis

[0174] Coomassie colloidal stained SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) was performed on a NU-PAGE gel from Novex(San Diego, Calif.) (4-12% MES, running buffer, sample buffer, and/ordithiothreitol, DTT reducing agent). The alum-adjuvanted powderformulations were reconstituted with water and centrifuged to remove thesupernatant. The alum pellet was re-suspended in 200 mM SodiumPhosphate, pH 7 with 0.1% SDS. The liquid suspension was then mixed withsample buffer from the Novex gel kit. The cocktail samples were thenheated at 95 ° C. for 5 minutes and vortexed prior to loading on thegel. The gels were run for 35 minutes at 200V/120 mA/25W using a NovexPowerEase 500 power supply, and then coomassie stained (Novex ColloidalBlue Stain) and destained with water. A gel image was scanned on aBioRad gel scanner (Model GS-700 Imaging Densitometer). The scanner wasequipped with quantitation software (Quantity One) that can quantify theintensity of the gel bands. The unit of signal intensity is OpticalDensity (O.D.). All samples were compared against a molecular weightmarker (Mark 12, Novex).

[0175] The results of both non-reducing and reducing gels are presentedin FIG. 4. As can be seen, no significant differences between theconcentrated liquid sample and the SFD samples were observed, showingthat the spray-freeze-drying process of the present invention does notaffect the antigen quality.

EXAMPLE 2 Spray Freeze Drying (“SFD”) of Biopharmaceutical (Protein)Compositions

[0176] 2.1 Objectives:

[0177] To assess the SFD process for use in preparing biopharmaceutical(protein pharmaceutical) powders and to further assess the powderedformulations with respect to the following criteria. (a) particle tapdensity); (b) particle size distribution; and (c) physical stability ofthe particles.

[0178] A model protein, bovine serum albumin (BSA), was used as thebasis for this study on SFD protein powders produced according to theinstant invention, and the evaluation of these powders based on physicalproperties, including particle size, tap density, and physicalappearance in terms of hygroscopicity and powder flowability. Anotherimportant aspect of the process that was considered was thefreeze-drying condition/cycle. Aggressive drying cycles were attemptedfor two purposes, shortening the drying time and applying higher dryingtemperatures to facilitate particle collapse during drying.

[0179] Spray-freeze-drying allows atomised formulation droplets to beimmediately frozen in liquid nitrogen and then freeze dried. The drypowder has a controlled particle size, and the shape of the particle isspherical. This process is highly efficient and has been demonstrated ashaving as benign effect on biopharmaceuticals such as proteins andpeptides. A number of formulation compositions were assessed for theiraffect on resultant particle density, wherein a range of suitableexcipient compositions permits particles of acceptable characteristicsto be prepared from widely different proteins and peptides. A typicalformulation is the combination of an amorphous sugar to provide proteinstability, a crystalline component to render particle strength, and apolymer to offer particle integrity. These excipients must be highlywater soluble to achieve a high solid content in the liquid formulation,a parameter that is essential to producing particles of acceptabledensity. The selection of the particular excipient combination muststrike a balance between hygroscopicity of the resulting powder and theshort-term and long-term physical stability of the biopharmaceuticalguest. Accordingly, these criteria were also applied during theevaluation of the various excipient compositions used in the instantstudy. All of the excipients used in the study are consideredparenterally acceptable and approvable for use in humans.

[0180] 2.2 Theoretical Considerations of Ultrasonic Atomisation:

[0181] Since the atomisation process is an important pre-requisite toattain a fairly narrow particle size distribution, some basictheoretical considerations were addressed.

[0182] In simple terms ultrasonic atomisation process results frominstabilities created in the liquid capillary waves that form on theatomising surface as a result of the energy provided to the surface inthe form of high frequency vibrations. Drops are then formed when theultrasonic energy exceeds the liquid's surface tension. The sizedistribution of the drops depends principally on the frequency of thenozzle, as well surface tension and specific gravity, although these areminor factors. Equation 2.1 relates the number median diameter to thefrequency, specific gravity and surface tension;

d _(N,0.5)=0.34(8πs/pf ²)^(1/3)   (2.1)

[0183] wherein s is the surface tension of the liquid, p is the specificgravity and f is the frequency. Thus from Equation 2.1 it can be seenthat frequency is the principal factor in determining drop size as itenters the equation as a square, whilst surface tension and the specificgravity only enter as the first power.

[0184] The principal consequence of Equation 2.1 is that the dropletdiameter is proportional to f^(2/3). This implies that higher operatingfrequencies produce smaller droplets.

[0185] 2.3 Spray Freeze Drying (SFD) Apparatus and Methods:

[0186] 2.3.1 The SFD Apparatus

[0187] The SFD apparatus featured an ultrasonic atomizer (Sono-TekCorporation, Milton, N.Y.) having a spray nozzle (Model #05793) and apower supply (Model #06-05108). The nozzle was equipped with aquasi-electric quartz crystal capable of vibrating at a specificfrequency that determines the size of the droplets. Ultrasonic nozzlesof 60 and 48 kHz frequencies were used. Circular metal pans (16-cm indiameter by 6-cm in height) were used to contain the liquid nitrogen.For the lyophilization, a shelf freeze dryer (Model #TDS2C2B5200,Dura-Stop, FTS System, Stone Ridge, N.Y.) was used. This dryer can holdsix metal pans in one batch run. Other apparatus included a magneticstirrer with magnetic stir bars, and a peristaltic pump (Model#77120-70, MasterFlex C/L, Barnant Company, Barrington, Ill.). Aschematic of the Spray Freeze Drying Process is depicted in FIG. 5.

[0188] 2.3.2 The SFD Methods

[0189] The liquid protein formulation was delivered by the peristalticpump (flow rate of 1-5 mL/min) into the ultrasonic atomiser (48 or 60kHz). The liquid formulation was then sprayed into a liquidN₂-containing pan. After spraying, the pan containing the frozenparticles in liquid nitrogen was transferred to a pre-cooled freezedryer (−55° C.). Liquid nitrogen will then evaporate. Four sets offreeze-drying conditions that were used in this study are listed belowin Tables 2.1 - 2.4. TABLE 2.1 Freeze-drying cycle (conservative 48-hourcycle) Stage/ Cycle Conditions Freezing pre-cool shelf temperature (ST)= −50° C. ramp at 2.5° C./min to ST = −55° C., hold for 15 min wait forproduct temp (PT) = −48° C., hold for 15 min Primary condenser/vacuum(C/V) switched “on” Drying when condenser temp. reaches −40° C., vacuumpump turned on wait for chamber vacuum to reach 150 mT wait for forelinevacuum to reach 100 mT ramp at 1.0° C./min to ST = −10° C., hold for 24hours Secondary ramp at 1.0° C./min to ST = 20° C., hold for 24 hoursDrying

[0190] TABLE 2.2 Freeze-drying cycle (semi-aggressive 20-hour cycle)Stage/ Cycle Conditions Freezing pre-cool shelf temperature (ST) = −50°C. ramp at 2.5° C./min to ST = −55° C., hold for 15 min wait for producttemp (PT) = −48° C., hold for 15 min Primary condenser/vacuum (C/V)switched “on” Drying when condenser temp. reaches −40° C., vacuum pumpturned on wait for chamber vacuum to reach 150 mT wait for forelinevacuum to reach 100 mT ramp at 1.0° C./min to ST = −10° C., hold for 5hours ramp at 1.0° C./min to ST = 0° C., hold for 5 hours Secondary rampat 1.0° C./min to ST = 15° C., hold for 5 hours Drying ramp at 1.0°C./min to ST = 25° C., hold for 5 hours

[0191] TABLE 2.3 Freeze-drying cycle (aggressive 20-hour cycle) Stage/Cycle Conditions Freezing pre-cool shelf temperature (ST) = −50° C. rampat 2.5° C./min to ST = −55° C., hold for 15 min wait for product temp(PT) = −48° C., hold for 15 min Primary condenser/vacuum (C/V) switched“on” Drying when condenser temp. reaches −40° C., vacuum pump turned onwait for chamber vacuum to reach 150 mT wait for foreline vacuum toreach 100 mT ramp at 1.0° C./min to ST = −10° C., hold for 10 hoursSecondary ramp at 1.0° C./min to ST = 15° C., hold for 5 hours Dryingramp at 1.0° C./min to ST = 25° C., hold for 5 hours

[0192] TABLE 2.4 Freeze-drying cycle (most aggressive 16-hour cycle)Stage/ Cycle Conditions Freezing pre-cool shelf temperature (ST) = −50°C. ramp at 2.5° C./min to ST = −55° C., hold for 15 min wait for producttemp (FT) = −48° C., hold for 15 min Primary condenser/vacuum (C/V)switched “on” Drying when condenser temp. reaches −40° C., vacuum pumpturned on wait for chamber vacuum to reach 150 mT wait for forelinevacuum to reach 100 mT ramp at 1.0° C./min to ST = −5° C., hold for 6hours Secondary ramp at 1.0° C./min to ST = 25° C., hold for 10 hoursDrying

[0193] After drying, the powder-containing pan was transferred into adry box purged with nitrogen to maintain a relative humidity of <30%.

[0194] 2.4 Powder Characterization:

[0195] 2.4.1 Particle Size Analysis

[0196] The mean geometric diameter of the particles in the volumedistribution can be determined using an AccuSizer 780 (Particle SizingSystems, Santa Barbara, Calif.). Based on the light obscurationtechnique, AccuSizer determines the particle size distribution withoutassuming the shape distribution of the particle. In addition, the sizeof the particle population between 10% and 90% (volume) can also bedetermined. Each analysis required approximately 5 mg of the powdersample. Powder samples are suspended in light mineral oil and sonicatedfor 5-10 seconds to remove agglomeration of particles before analysis.

[0197] The mean aerodynamic diameter of the particles in the volumedistribution was determined using a dry powder dispersion-based particlesize analyser (Aerosizer, API). In addition, the size of the particlepopulation between 10% and 90% (in volume distribution) was alsoreported for each particle size distribution. Each analysis requiredapproximately 5 mg of the powder sample.

[0198] 2.4.2 Image Analysis

[0199] Visual analysis of the particles was performed using an opticalmicroscope (Model DMR, Leica, Germany) with 10×-eyepiece lens and10×-objective lens. The system was equipped with a Polaroid camerasystem for image output. Digital images were captured and stored.

[0200] 2.4.3 Tap Density

[0201] Each powder sample was weighed in a glass vial and gently tappedagainst the lab bench for 20 times. By visual inspection, water of anequivalent volume to that of the powder was placed into an empty vial ofthe same type. The tap density of the powder sample could be calculatedby dividing the powder sample weight with the water sample weight(assuming water density=1 g/mL).

[0202] 2.4.4 Moisture Content Analysis

[0203] Approximately 4-5 mg of the powder sample was recovered from thetrilaminate cassettes and transferred into an aluminum weighing-vesseland the weight was recorded. The sample was loaded into a Karl FisherCoulometer (Model 737, Brinkmann) equipped with a drying oven (Model707). The sample was heated to 150 ° C. for 150 sec with a gas flow rateof 100 mL/min within the drying oven. Sample extraction time was 120seconds.

[0204] 2.5 Pharmaceutical Formulations and Results:

[0205] In the series of experiments discussed herein below, variousformulations were tested wherein two excipients were kept constant,namely mannitol and trehalose. Trehalose was selected primarily forstabilizing the protein during lyophilization and long-term storage.Mannitol was added to the formulation to impart rigidity to theparticles, as mannitol is a crystalline material.

[0206] The powder formulations that were produced using the SFD processof the present invention and the particular apparatus and methodsdescribed above. In the first series of experiments, formulationscontaining polyvinylpyrrolidone (PVP) were assessed. PVP is aparenterally acceptable excipient and imparts plasticity to theformulation, and hence was selected as a preferred bulking agent.

[0207] 2.5.1 PVP Formulations

[0208] In the following series of experiments, the objective was todetermine the particle physical properties when the concentration of thePVP bulking agent was altered from 18 to 36% w/w. The particularformulations tested are reported below in Table 2.5. TABLE 2.5 SFD PVPformulations Solids Nozzle Freeze Batch Formulation Composition ContentFreq. Drying Number (% w/w) (% w/w) (kHz) Cycle 156-16-1 10% BSA, 45%trehalose, 35 60 See 27% mannitol, 18% PVP Table (K17) and 0.1% Pluronic2.1 F68. 156-16-2 10% BSA, 44.9% 35 48 See trehalose, 26.9% mannitol,Table 18% PVP (K17), 0.1% 2.1 methionine and 0.1% Pluronic F68 156-16-310% BSA, 26.9% 35 60 See trehalose, 26.9% mannitol, Table 35.9% PVP(K17), 0.1% 2.1 methionine and 0.1% Pluronic F68. 156-16-4 10% BSA,26.9% 35 48 See trehalose, 26.9% mannitol, Table 35.9% PVP(K17), 0.1%2.1 methionine and 0.1% Pluronic F68.

[0209]

[0210] Image Analysis Results:

[0211] Photomicrographs (FIGS. 6a-6 d) of the SFD formulations definedin Table 2.5 reveal that the particles are of a spherical morphology,with a fairly uniform particle size. There are some particles that areagglomerated, which is a consequence of PVP present in the formulations.PVP is an effective binder, and formulations containing this excipientwill have some tendency to agglomerate due to the adhesive nature of thepolymeric excipient.

[0212] Particle Size Results:

[0213] As seen below in Table 2.6, the mean particle sizes of the PVPformulations were similar, with the exception of batch number 156-16-3,which had a smaller mean particle size. Comparison of batches 156-16-1and 156-16-3, which were both manufactured using a 60 kHz ultrasonicnozzle (the only difference between the two formulations being the ratioof the excipients), reveals that the 3:3:4 excipient ratio producedsmaller particles. Similarly, comparison of batches 156-16-2 and156-16-4 showed the same trend, although the effect was less marked.TABLE 2.6 Particle size results Mean Size Median size Batch number (μm)D_(0.10)-D_(0.90) (μm) 156-16-1 39.2 ± 1.4 24.9-59.3 40.0 156-16-2 40.4± 1.4 26.8-60.3 40.8 156-16-3 36.3 ± 1.3 24.7-52.7 36.6 156-16-4 39.6 ±1.4 26.3-58.9 39.9

[0214] Moisture Content Analysis:

[0215] The Karl Fischer results presented in Table 2.7, below, revealthat the moisture content of all of the PVP formulations was <3%. Theseresults further indicate that the ratio of excipients 3:3:4 produced adrier product. TABLE 2.7 Karl Fischer (moisture content) results BatchNumber % Moisture 156-16-1 2.9 156-16-2 2.9 156-16-3 1.9 156-16-4 2.4

[0216] Particle Density Results:

[0217] As can be seen by the results reported in Table 2.8 below, thetap densities of all four PVP formulations were similar and withinacceptable ranges. TABLE 2.8 Tap density Tap Density Batch Number(g/cm³) 156-16-1 0.65 156-16-2 0.66 156-16-3 0.64 156-16-4 0.67

[0218] 2.5.2 Various Sugar Formulations

[0219] In the following series of experiments, the objective was todetermine the particle physical properties using differing combinationsof sugars (raffinose, sucrose) and other common excipients (glycine).The particular formulations tested are reported below in Table 2.9.TABLE 2.9 SFD sugar formulations Ultrasonic Formulation Solids NozzleFreeze Batch Composition Content frequency Drying Number (% w/w) (% w/w)(kHz) Cycle 156-35-1 10% BSA, 36% raffinose, 35 60 See 27% trehalose,27% Table mannitol. 2.2 156-35-2 10% BSA, 36% raffinose, 35 60 See 36%mannitol and 18% Table PVP (K17). 2.2 156-35-3 30% BSA, 40% raffinose 3560 See and 30% mannitol. Table 2.2 156-42-1 10% BSA, 36% raffinose, 3560 See 27% trehalose, 27% Table mannitol. 2.3 156-42-2 10% BSA, 27%raffinose, 35 60 See 27% mannitol 18% Table glycine and 18% 2.3trehalose. 156-42-4 10% BSA, 27% raffinose, 35 60 See 27% sucrose and36% Table mannitol. 2.3

[0220] Image Analysis Results:

[0221] As can be seen in FIGS. 7a-7 f, the particles prepared by the SFDprocess from the formulations defined in Table 2.9 have a sphericalmorphology, with a fairly uniform particle size. However, batch numbers156-35-1, 156-35-2 and 156-35-3 (FIGS. 7a, 7 b and 7 c, respectively)appear to have a few oversize particles. Batch number 156-42-2 (FIG. 7e)had an irregular morphology and appears to be in the process ofdeliquescence, suggesting the highly hygroscopic nature of theformulation.

[0222] Particle Size Results:

[0223] The particle size results of the formulations of Table 2.9 arereported below in Table 2.10 and generally correspond to the estimatedsizes obtained from the photomicrographs. TABLE 2.10 Particle SizeResults Batch Mean Size Median size Number (μm) D_(0.10)-D_(0.90) (μm)156-35-1 39.5 ± 1.3 27.4-55.3 40.1 156-35-2 38.9 ± 1.3 26.0-55.7 39.8156-35-3 34.4 ± 1.4 22.7-50.0 35.3 156-42-1 34.4 ± 1.3 23.3-49.5 35.1156-42-2 36.1 ± 1.3 26.4-48.8 36.4 156-42-4 38.8 ± 1.3 27.4-52.9 40.0

[0224] Moisture Content Analysis:

[0225] The Karl Fischer results presented in Table 2.11, below, revealthat the moisture content of all of the tested formulations was <5%. Ascan be seen, batch number 156-35-2 had the lowest residual moisture,likely due to the increased amount of mannitol in that formulation.TABLE 2.11 Karl Fischer (moisture content) results Batch Number %Moisture 156-35-1 4.8 156-35-2 3.0 156-35-3 4.2

[0226] Particle Density Results:

[0227] As can be seen by the results reported in Table 2.12, below, thetap densities of all of the tested formulations were similar and withinacceptable ranges. As can also be seen, the addition of glycine toformulation for batch number 156-42-2 caused a marked increase in tapdensity relative to the other formulations. The variables that can altertap density of a powder are multifaceted and depend principally on theparticle size, particle size distribution, crystal habit and rugosity.Alterations in these variables by introduction of another excipient oran increase in the amount of excipient/active formulation will lead topredictable differences in particle tap densities.

[0228] The use of raffinose as a bulking agent in these formulationsresulted in particles of acceptable physical characteristics, and therewas a lower incidence of agglomeration when compared to the PVPformulations. TABLE 2.11 Tap density Tap Density Batch Number (g/cm³)156-35-1 0.65 156-35-2 0.57 156-35-3 0.59 156-42-1 0.68 156-42-2 0.75156-42-4 0.67

[0229] 2.5.3 Various Dextran Formulations

[0230] In the following series of experiments, the objective was todetermine the particle physical properties using dextran as the bulkingagent. Dextran forms a 10 glass having a high glass transitiontemperature (TG). The particular dextran-containing formulations testedare reported below in Table 2.13. TABLE 2.13 SFD dextran formulationsNozzle Solids fre- Freeze Batch Formulation Composition Content quencyDrying Number (% w/w) (% w/w) (kHz) Cycle 156-35-4 10% BSA, 27%trehalose, 35 60 See 27% mannitol and 36% Table dextran (10 kDa). 2.2156-42-3- 10% BSA, 27% trehalose, 35 60 See 1 27% mannitol and 36% Tabledextran (10 kDa). 2.3 156-42-3- 10% BSA, 27% trehalose, 35 60 See 2 27%mannitol and 36% Table dextran (10 kDa). 2.3 156-53-1 10% BSA, 27%trehalose, 35 60 See 27% mannitol and 36% Table dextran (10 kDa). 2.4156-61-1 10% BSA, 27% trehalose, 35 60 See 27% mannitol and 36% Tabledextran (10 kDa). 2.4 156-65-1 10% BSA, 36% trehalose, 40 60 See 18%mannitol, 18% arginine Table glutamate, 18% dextran (10 2.4 kDa).

[0231] Image Analysis Results:

[0232] As can be seen in FIGS. 8a-8 f, the SFD dextran formulations(defined in Table 2.13) provided particles with a spherical morphology,a narrow size distribution and there was also a noticeable lack ofagglomerated particles.

[0233] Particle Size Results:

[0234] The particle size results from the assessment of the variousdextran batches (the formulations of Table 2.13) are reported hereinbelow in Table 2.14, and generally correspond to the estimated sizesobtained from the photomicrographs. As can be seen, there was a markedincrease in the mean particle size of batch number 156-53-1 whencompared to batch number 156-42-3-1, but this can be explained by thePSD generated by the Aerosizer, which was skewed more to the right, whencompared with the PSD of batch number 156-42-3-1, which is indicative oflarge particles. Batch number 156-61 -1, which is a scaled-upformulation of batch number 156-53-1 yielded a similar PSD. Batchesnumbers 156-53-1 and 156-42-3-1 have similar BSA and excipientcompositions, however a freeze-drying cycle of 16 hours was utilised forthe former.

[0235] These Aerosizer data also reveal that there was a marked increasein the mean particle size when using a shorter drying cycle, howeverinspection of FIGS. 8c and 8 f does not show any significant differencesin terms of morphology or size. From these data it can be postulatedthat a longer drying cycle results in a more collapsed particle and thismay have an effect on the intra-particle porosity, in which case thiswould affect the particle size as measured by the Aerosizer as theaerodynamic size depends on intraparticle porosity. Batch number156-65-1 had the largest mean particle size. This can be attributed tothe increase in solids content relative to the other dextranformulations of Table 2.13. TABLE 2.124 Particle size results Mean SizeMedian size Batch number (μm) D_(0.10)-D_(0.90) (μm) 156-35-4 33.1 ± 1.323.3-46.1 33.6 156-42-3-1 35.0 ± 1.3 24.7-49.1 35.4 156-42-3-2 33.6 ±1.3 23.4-47.3 34.2 156-53-1 39.2 ± 1.4 26.2-57.6 39.9 156-61-1 39.7 ±1.4 25.4-61.9 40.1 156-65-1 45.3 ± 1.3 30.8-64.9 46.5

[0236] Particle Density Results:

[0237] As can be seen by the results reported in Table 2.15, below, thetap densities of all of the tested dextran formulations were similar andwithin acceptable ranges. A review of these densities also indicatesthat the freeze-drying time had a negligible overall effect on the tapdensity.

[0238] The marked increase in the mean particle size of batch number156-53-1, when compared to batch number 156-42-3-1, can not be due tothe production of a more collapsed particle as the tap densities andlight microscopy photomicrographs would have indicated a differencebetween the two batches, hence the difference between the mean particlesizes of the two batches is attributed to sampling variability. TABLE2.13 Tap density Tapped Density Batch Number (g/cm³) 156-35-4 0.54156-42-3-1 0.57 156-42-3-2 0.56 156-53-1 0.53 156-61-1 0.56 156-65-10.61

[0239] 2.5.4 Various Salt Formulations

[0240] In the following series of experiments, the objective was todetermine the particle physical properties of formulations incorporatingdiffering combinations of a salt bulking agent (arginine glutamate) andother common excipients (alanine, Pluronic, methionine). The particularformulations tested are reported below in Table 2.16. TABLE 2.146 SFDsalt formulations Nozzle Fre- Freeze Batch Formulation CompositionSolids quency drying Number (% w/w) Content (kHz) Cycle 156-57-1 10%BSA, 36% trehalose, 35 60 See 36% mannitol, 18% alanine. Table 2.3156-57-2 10% BSA, 27% trehalose, 35 60 See 27% mannitol 36% arginineTable 2.3 glutamate. 156-65-2 10% BSA, 36% trehalose, 40 60 See 18%mannitol, 36% arginine Table 2.4 glutamate. 156-71-1 10% BSA, 36%trehalose, 40 60 See 18% mannitol, 36% arginine Table 2.1 glutamate.156-76-1 10% BSA, 35.9% trehalose, 35 60 See 18% mannitol, 35.9% Table2.4 arginine glutamate, 0.1% Pluronic F168 and 0.1% methionine. 156-76-210% BSA, 26.9% trehalose, 35 48 See 26.9% mannitol, 35.9% Table 2.4arginine glutamate, 0.1% Pluronic F168 and 0.1% methionine.

[0241] Image Analysis Results:

[0242] As can be seen in FIGS. 9a-9 f, the SFD salt formulations definedin Table 2.16, provided particles with a spherical morphology and narrowsize distribution. FIG. 9a is a photomicrograph of batch number156-57-1, which shows that the particles have agglomerated after thefreeze drying process, moreover the particles shown in FIG. 9a have athick rounded edge, which is indicative of deliquescence. The other saltformulations do not show any evidence of agglomeration.

[0243] Accordingly, X-ray powder diffraction (XRPD) was conducted on thearginine glutamate and a spray freeze dried formulation containing theaforementioned excipient. This was conducted to elucidate the morphologyof the excipient prior to freeze drying and after freeze drying.Analysis of the XRPD pattern for arginine glutamate prior to freezedrying showed distinct peaks, which is indicative of a crystallinematerial. Analysis of the XRPD pattern for batch number 156-76-1 showeda diffuse halo, suggesting an amorphous formulation.

[0244] Particle Size Results:

[0245] The particle size results of the various salt formulations (theformulations of Table 2.16) are reported herein below in Table 2.17, andgenerally correspond to the estimated sizes obtained from thephotomicrographs. TABLE 2.15 Particle size results Mean Size Median sizeBatch number (μm) D_(0.10)-D_(0.90) (μm) 156-57-1 40.3 ± 1.3 27.8-57.640.8 156-57-2 37.7 ± 1.4 23.7-58.0 38.3 156-65-2 44.1 ± 1.4 28.2-66.145.4 156-71-1 41.3 ± 1.4 26.0-63.5 42.7

[0246] Particle Density Results:

[0247] As can be seen by the results reported in Table 2.18, below, thetap densities of all of the tested formulations were relatively high,with the single exception of batch number 156-76-1, and all withinacceptable ranges. These results demonstrate that the use of arginineglutamate as the bulking agent in the formulations of the presentinvention provides particles of acceptable physical characteristics,however, the use of alanine was not deemed optimal due the deliquescenceof the formulation. TABLE 2.16 Tap density Batch Number Tapped Density(g/cm³) 156-57-1 0.67 156-57-2 0.69 156-65-2 0.63 156-71-1 0.66 156-76-10.46 156-76-2 0.57

[0248] 2.5.5 Further Salt Formulations

[0249] As with the above series of experiments, the objective of thisseries of experiments was to determine the particle physical propertiesof formulations incorporating combinations of different alternativebulking agents (arginine aspartate) and other common excipients(Pluronic F 168, methionine, Tween 80). The particular formulationstested are reported below in Table 2.19. TABLE 2.179 SFD formulationsNozzle fre- Freeze Batch Formulation Composition Solids quency dryingNumber (% w/w) Content (kHz) cycle 156-80-1 10% BSA, 27% trehalose, 3560 See Table 27% mannitol, 36% 2.4 arginine aspartate. 156-80-2 10% BSA,5% Pluronic 35 60 See Table F168, 59.5% trehalose 2.4 and 25.5%mannitol. 156-80-3 10% BSA, 35.9% trehalose, 40 60 See Table 18%mannitol, 35.9% 2.4 arginine glutamate, 0.1% methionine and 0.1% Tween80.

[0250] Image Analysis Results:

[0251] As can be seen in FIGS. 10a-10 c, the SFD salt formulationsdefined in Table 2.19 produced particles with a spherical morphology.However, a number of oversize particles are evident, particularly inbatch number 156-80-3. In addition, batch number 156-80-2 (FIG. 10b) hadparticles that seemed to be fused together, likely as a consequence ofthe high Pluronic content in the formulation.

[0252] Particle Size Results:

[0253] The particle size results of the various salt formulations (theformulations of Table 2.19) are reported herein below in Table 2.20, andgenerally correspond to the estimated sizes obtained from thephotomicrographs depicted in FIGS. 10a-10 c. Batch number 156-80-1yielded the smallest mean particle size, due to the lower solids contentused, whilst batch number 156-80-2 yielded the largest particle sizewhich was partially as a consequence of particle agglomeration/fusion(see FIG. 10b). TABLE 2.20 Particle size results Mean Size Median sizeBatch number (μm) D_(0.10)-D_(0.90) (μm) 156-80-1 33.6 ± 1.4 22.5-48.434.6 156-80-2 41.8 ± 1.4 26.2-63.2 43.4 156-80-3 37.7 ± 1.4 24.9-55.938.6

[0254] Particle Density Results:

[0255] As can be seen by the results reported in Table 2.21, below, thetap densities of all of the tested formulations were relatively high andwithin acceptable ranges. The formulation of batch number 156-80-1 had arelatively lower density due to a lower starting solids content (35%).These results demonstrate that the use of arginine aspartate as thebulking agent in the formulations of the present invention providesparticles of acceptable physical characteristics. TABLE 2.21 Tap densityBatch Number Tapped Density (g/cm³) 156-80-1 0.51 156-80-2 0.72 156-80-30.63

EXAMPLE 3 Spray Freeze Drying of Alum-Adjuvanted Vaccine Compositions

[0256] 3.1 Objectives:

[0257] To assess the SFD process for use in preparing alum-adjuvantedvaccine powders and to further assess the powdered formulations withrespect to their in vivo performance using epidermal powder immunization(“EPI”) and conventional needle and syringe administration techniques.Hepatitis B vaccine (Alum-HBsAg) and a diphtheria/tetanus toxoid vaccine(Alum-DT) were selected for the studies since the Alum-HBsAg compositioncontains aluminium hydroxide adjuvant and the Alum-DT compositioncontains aluminium phosphate adjuvant.

[0258] 3.2 Materials:

[0259] The chemicals and excipients that were used to produce thevarious vaccine compositions used in this study are summarized in Table3.1 below. All alum formulations were concentrated by centrifugation(Allergra 6R Centrifuge, Beckman Instrument, Palo Alto, Calif.) prior touse. TABLE 3.1 Chemicals/excipients used in the study. Chemical Lot #Source Comment Aluminum 8934 Accurate Manufactured by HCI phosphateChemical and Biosector (Adjus-Phos, Scientific (Frederikssund, 2% AlPO₄)(Westbury, NJ) Denmark) Aluminum Accurate Manufactured by hydroxideChemical and Superflos Biosector (Alhydrogel, Scientific (Vedbaek,Denmark) 3% Al(OH)₃) Diphtheria G9334 Accurate Manufactured by toxoid(dT, MW Chemical and Statens Serum Institute, 58 kDa) ScientificDenmark, and provided at 5 mg/mL (1 Lf = 2.42 μg), used as supplied.Tetanus toxoid G9486 Accurate Manufactured by (tT, MW 150 Chemical andStatens Serum Institute, kDa) Scientific Denmark, and provided at 2mg/mL (1 Lf = 2.44 μg), used as supplied. Alum phosphate- CSL LimitedBulk containing 5 adjuvanted DT (Parkville, w/v % alum phosphateAustralia) adsorbed with both dT and tT at 563 Lf/mL Alum hydroxide-Rhein 20 μg HBsAg adsorbed adjuvanted Amaericana to 0.5 mg of aluminumhepatitis-B S.A. (Buenos or 1.5-mg of aluminum surface antigen Ares,hydroxide. (HBsAg) Argentina) Dextran (MW 18H0568 Sigma (St. Reagentgrade, used as 37,500 Da) Louis, MO) supplied Glycine 28H0103 SigmaReagent grade, used as supplied Mannitol 127H0960 Sigma Reagent grade,used as supplied Trehalose 28H3797 Sigma Reagent grade, used asdihydrate supplied

[0260] 3.3 Methods:

[0261] 3.3.1 Spray-Freezing (SF) and Spray-Freeze-Drying (SFD)

[0262] Liquid formulations were delivered by a peristaltic pump (Model#77120-70, MasterFlex C/L, Barnant Company, Barrington, Ill.) at a flowrate of 2.0 mL/min into an ultrasonic atomizing system (Sono-TekCorporation, Milton, N.Y.) consisting of a spray nozzle (Model #05793)and a power supply (Model #06-05108). The nozzle is equipped with aquasi-electric quartz crystal capable of vibrating at a specificfrequency that determines the size of the droplets. A 60 kHz sprayingnozzle produces droplets mostly within the range of 20-80 μm. Atomizeddroplets were sprayed into a liquid N₂-containing pan (16 cm in diameterby 6 cm in height). For formulations subjected to thespray-freezing/thawing experiment, the frozen powder was transferred toa glass vial and thawed at ambient conditions. For frozen dropletsundergoing drying, the pan containing frozen particles in liquidnitrogen was transferred to a pre-cooled (−55° C.) shelf freeze dryer(Model #TDS2C2B5200, Dura-Stop, FTS System, Stone Ridge, N.Y.). Theliquid nitrogen evaporated in a few minutes. The freeze-drying conditionwas set at −25° C. for 18 hours and 20 ° C. for 10 hours. The rampingrate was 1° C./minute consistently. The vacuum pressure was 100 mlthroughout the cycle. After drying, the powder-conaining pans weretransferred into a dry box purged with nitrogen (at <30% relativehumidity) for powder collection. The same lyophilization cycle was usedfor liquid formulations without SF with freezing achieved by storing ina −20° C. freezer overnight.

[0263] 3.3.2 Powder Formation by Compress/Grind/Sieve (C/G/S) Method

[0264] To prepare powders of high density for the epidermal powderimmunization (EPI) study, freeze-dried (FD) and SFD formulations werecompressed in a stainless steel dye of 13-mm in diameter (Carver Press,Wabash, IN) at a pressure of 12,000-15,000 pounds for 5-10 minutes. Thecompressed discs were ground manually using a mortar and pestle, andthen the ground powder was manually sieved through a stack of 3-insieves (Fisher Scientific Products, Pittsburgh, PA) of four sizes, 20,38, 53, and 75 μm.

[0265] 3.3.4 Spray-Drying (SD)

[0266] A bench-top mini spray dryer (Buchi B-191, Brinkmann, Westbury,N.Y.) was used to prepare placebo alum formulations. Using compressedair from an in-house supply (˜80 psi), a two-fluid nozzle (0.5 mm)atomized the aqueous feed solution. The standard operating conditionswere: inlet air temperature of 130° C., drying air blown at the fullscale, atomizing air flow rate of 500 L/hr, and liquid feed rate of 10mL/min. This condition resulted in an outlet air temperature of 70° C.

[0267] A laboratory spray dryer (Mobile Minor, Niro A/S, Soeborg,Denmark) was used to prepare Alum-DT formulation with the followingconditions. The two-fluid nozzle was operated at an atomizing pressureof 2 bar. The inlet air temperature was set at 160° C. drying air withfull-blown drying air. As the liquid was fed at 30 mL/min, the airoutlet temperature measured at 65-70° C.

[0268] 3.3.5 Air-Drying (AD)

[0269] Liquid alum-adjuvanted vaccine formulations were placed in apolystyrene weigh boat and allowed to dry overnight under the ambientconditions. Gentle agitation by a magnetic bar stirring was appliedthroughout the process to minimize phase separation.

[0270] 3.3.6 Optical Microscopy

[0271] Visual analysis of the particles was performed using an opticalmicroscope (Model DMR, Leica, Germany) with 10×-eyepiece lens and10×-objective lens. The system was equipped with a Polaroid camerasystem for image output.

[0272] 3.3.7 Particle Size Analysis

[0273] The mean geometric/aerodynamic diameter of the particles in thevolume distribution was determined using a time-of-flight particle sizeanalyzer (Aerosizer, API, Minneapolis, Minn.). The mean volumetric sizewas calculated by the software using the density of 1.0 and particlepopulation between 10% (D₁₀) and 90% (D₉₀) was reported for particlesize distribution. Each analysis requires approximately 3-5 mg of thepowder sample. For liquid suspensions, the particle size distributionwas measured using a light obscuration-based particle size analyzer(AccuSizer 780, Particle Sizing Systems, Santa Barbara, Calif.).

[0274] 3.3.8 SDS-PAGE

[0275] Coomassie colloidal-stained SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) was performed on a Nu-PAGE gel from Novex(San Diego, Calif.) (4-12% MES, running buffer, sample buffer, and/orDithiothreitol reducing agent). The alum-adjuvanted powder vaccineformulations were reconstituted with water and centrifuged to remove thesupernatant. The alum pellet was re-suspended in 200 mM sodiumphosphate, pH 7 with 0.1% SDS. The liquid suspension was then mixed withsample buffer from the Novex gel kit. The cocktail samples were thenheated at 95° C. for 5 minutes and vortexed prior to loading on the gel.The gels were run for 35 minutes at 200V/120 mA/25W using a power supply(PowerEase 500, Novex), and then coomassie stained (Novex Colloidal BlueStain) and destained with water. The gel images were scanned on a gelscanner (Model GS-700 Imaging Densitometer, BioRad) equipped with aquantitation software (Quantity One), which can quantify the intensityof the gel bands. The unit of signal intensity is Optical Density(O.D.). All samples were compared against a molecular weight marker(Mark 12, Novex).

[0276] 3.3.9 EPI Using a PowderJect® Powder Injection Device

[0277] A PowderJect® powder injection device (needleless syringe) wasused to immunize hairless guinea pigs. The device is approximately 15 cmin length and includes a gas cylinder (5-ml volume), rupture chamber, atrilaminate particle cassette, a nozzle, and a silencer element. Thestainless steel gas cylinder is filled with medical grade helium gas to40-bar pressure. The trilaminate cassette (11-mm O.D., 6-mm I.D., and4-mm height) is constructed of a thick ethylene vinyl acetate washerwith rupture membranes heat sealed to each side within which thepowdered vacine sample is housed. The rupture membranes are formed froma thin film (20 μm) made of semi-transparent polycarbonate. Uponactuation, the helium gas is released from the gas cylinder and causespressure build-up in the rupture chamber. The escaping gas overcomes therupture strength of the rupture membranes, causing the membranes torupture, whereby the gas sweeps through the trilaminate cassette andpropels the vaccine powder as projectiles into the skin. The helium gasis reflected off the skin and exhausted through the silencer element.The depth of powder penetration was experimentally optimized to deliverpowders to the epidermal layer of the skin for optimal tolerance andmaximal efficacy.

[0278] 3.3.10 Mice Immunization and Serum Collection

[0279] Five to seven week-old female BALB/c mice (Harlen-Sprague-Dawley,Indianapolis, Ind.) were used to assess the immunogenicity of powderedalum-adsorbed hepatitis B vaccines. FD and SFD powder formulations werereconstituted with distilled water and administered by intraperitoneal(IP) injection using a 26⅕ needle. Each injection administered 200 μl ofsolution containing 2 μg of hepatitis B surface antigen adsorbed onalum. Control mice were immunized with the same dose of untreated liquidhepatitis B vaccine. A boost immunization was administered on day 28.

[0280] Blood was collected via retro-orbital bleeding under anaesthesiaprior to each vaccination and two weeks post boost.

[0281] 3.3.11 Guinea Pig Immunization and Serum Collection Hairlessguinea pigs (Charles River, Wilmington, Mass.) were used to assess theimmunogenicity of powder formulations of alum adsorbed diphtheria toxoid(dT) and tetanus toxoid (tT) following EPI. The gneral methods for EPIare described in detail herein above and in the art. Briefly, one mg ofthe powdered vaccine compositions being tested was dispensed into atrilaminate cassette. The cassette was inserted into the PowderJectpowder injection device at the time of immunization. The device wasplaced against the left inguinal skin of the animals and actuated byreleasing the compressed helium at 40-bar pressure from the gascylinder. Control animals were immunized with 0.20 mL of DT vaccine insaline by intramuscular (IM) injection using a 26½-gauge needle.

[0282] Blood was collected via the kerotid blood vessel prior to eachvaccination and two weeks post boost.

[0283] 3.3.12 ELISA

[0284] The antibody responses to diptheria toxoid (dT) and tetanustoxoid (tT) components of the Alum-DT vaccine and to the HBsAg antigencomponent of the Alum-HBsAg vaccine were determined using a modifiedELISA method. A 96-well plate (Costar, Fisher Scientific Products,Pittsburgh, Pa.) was coated with 0.1 μg of antigen (HBsAg, dT, or tT) in30 mM phosphate buffered saline (PBS), pH 7.4, per well overnight at 4°C. Plates were washed 3 times with tris-buffered saline (TBS), pH 7.4,containing 0.1% Brij-35, and incubated with test sera diluted in PBScontaining 5% dry milk for 1.5 hr. A standard serum, containing a knownlevel of antibodies to dT, tT, or HBsAg, was added to each plate andused to standardize the titer in the final data analysis. The plateswere then washed and incubated with biotin-labeled goat anti-mouseantibodies (1:8,000 in PBS, Southern Biotechnology Associate,Birmingham, Ala.) for 1 hr at room temperature. Finally, the plates werewashed and developed with TMB substrate (Bio-Rad Laboratories, Melville,N.Y.). The endpoint titers of the sera were determined by 4-parameteranalysis using the Softmax Pro 4.1 program (Molecular Devices,Sunnyvale, Calif.) and defined as the reciprocal of the highest serumdilution with an OD reading above the background by 0.1. A referenceserum with a predetermined titer was used on every plate to calibratethe titers and adjust assay-to-assay and plate-to-plate variation.

[0285] 3.4 Powder Characterization:

[0286] Current commercial alum-adjuvanted vaccines are formulated atapproximately 2 w/v % of aluminum salt in saline for injection.Accordingly, commercially available vaccine compositions were reviewedbefore and after freezing using an optical microscope. The results ofthe study are depicted in FIGS. 11a-11 d. Based on optical microscopy,the Adju-Phos adjuvant (2 w/v % placebo AlPO₄ gel) shows smooth andsandy texture without discernible particles. After freezing at -20° C.,the thawing gel develops immediately significant coagulation (see FIG.11a), but the same gel shows only slight aggregation (light dots) afterspray-freezing (see FIG. 11b). The same difference was observed for theAlhydrogel adjuvant (3 w/v % placebo Al(OH) ₃) after freezing at −20° C.(see FIG. 11c) and spray-freezing (see the dark particles in FIG. 11d).By appearance, large particles were visible in the gel solution that hadbeen frozen in the −20° C. freezer and they rapidly settled to thebottom of the container. In addition, the volume of the settled alumparticles was significantly greater than that of the starting gel. Allthese observations suggest that alum gels will coagulate after regularfreezing. Coagulated alum gels are not reversible even under mechanicalforce such as sonication or vortexing. On the other hand, the absence ofcoagulation with the spray-frozen gel confirms that extremely fastfreezing significantly reduces the coagulation tendency of alum gelregardless of its salt type even in the absence of bulking orstabilizing agents. After lyophilization of the frozen gels, the extentof coagulation of the reconstituted alum appears to be similar to thefreeze/thaw samples, suggesting that freezing is a primary cause of alumcoagulation.

[0287] 5 3.5 In vivo Performance:

[0288] 3.5.1 Effect of Alum Coagulation on Immunogenicity of Alum-HBsAg

[0289] A mouse model was used to test if the immunogenicity of theAlum-HBsAg vaccine composition would be affected by the dryingmethodology and the size of the coagulated particles. Table 3.2, below,summarizes the study. TABLE 3.2 Immunogenicity Study for Alum-HBsAgpowder formulations. Drying Process Group (n = 8) Formulation (particlesize) 1 Q FD 2 Q FD (<20 μm) 3 Q FD (38-53 μm) 4 Q FD (53-75 μm) 5 Q SFD(38-53 μm) 6 R SFD (38-53 μm) 7 Control not dried (liquid)

[0290] The formulations used in the study were as follows. FormulationQ:Alum hydroxide (3.0 w/v %)/mannitol (1.9 w/v %)/glycine (0.5 w/v%)/dextran (0.61 w/v %) in which the alum concentration was achieved bycombining the Alum-HBsAg with the placebo alum hydroxide gel.Formulation R: Alum hydroxide (0.6 w/v %)/mannitol (2.8 w/v %)/glycine(1.2 w/v %)/dextran (0.58 w/v %). Control: the commercial Alum-HBsAgproduct (Rhein Biotech) used as supplied by the manufacturer.

[0291] The SFD powders prepared from Formulations Q and R differ in alumsalt concentration. Powders for Groups #2-4 were prepared by compressingthe FD Formulation Q followed by grinding and then sieving to produce 3different particle size-fractions. All powdered compositions were fullycharacterized as described above in Examples 1 and 2. The opticalmicrographs of the reconstituted powders again suggested that the SFDpowder could be readily re-suspended in water while the FD formulationwas highly coagulated. Another characterization method involvedre-suspending the powder sample in water and subjecting tolight-scattering particle size analysis (AccuSizer). The results of theparticle size analysis are depicted in FIGS. 12a and 12 b. FIG. 12ashows the alum particle size distribution for the two SFD powdersfalling in the same particle size range as the starting gel, suggestingno detectable aggregated particles. However, the reconstitutedFD/compress/grind/sieve powder (38-45 μm, Group #3 in Table 3.2) shows aparticle size range of 5-50 μm with a peak at 45 μm (FIG. 12b), whichoverlaps with the size of dry particles before rehydration.

[0292] In order to assess stability of the Alum-HBsAg compositions upondrying, SDS-PAGE analysis was performed under both non-reducing andreducing conditions. The results from the optical density scans of thenon-reduced SDS-PAGE analysis are reported below in Table 3.3. TABLE 3.3Optical density of HBsAg band (non-reduced SDS-PAGE) Light IntensityFormulation (O.D.) Formulation Q, FD 847 Formulation Q FD (particle 1235size <20 μm) Formulation Q, FD (particle 1021 size 53-75 μm) FormulationQ, SFD 1507 (particle size 38-53 μm) Formulation R, SFD 1479 (particlesize 38-53 μm) Control (liquid) 1671

[0293] In its native state, HBsAg is a highly aggregated particle of 22nm in diameter. No formulations dissociated into monomeric form underthe non-reducing condition. However, the light intensity of the singleband at the top of the SDS-PAGE gel differs among the formulations(Table 3.3). For example, the light intensity for the two SFDformulations is slightly lower than that for the control (startingliquid formulation). The FD formulation showed the lightest band whileband intensity increased when the FD formulation was formulated intoparticles using C/GIS. Interestingly, band intensity increased withdecreasing particle size. This observation appears to be related to alumgel coagulation since the antigen desorption from the surface ofaggregated alum particles may be restricted or blocked. Grinding the FDalum gel generated new surfaces exposing more antigen. The powder'sspecific surface area increases as the particle size decreases. Underreducing conditions, HBsAg particles were reduced to monomers ofapproximately 24 kDa. There was no difference observed in band patternand intensity among all the formulations. This is probably due to theease with which monomeric HBsAg can diffuse out of the tightly packedalum aggregates.

[0294] Immunogenicity of the various Alum-HBsAg formulations describedin Table 3.2 above was tested in the mouse model. The dose of HBsAgadministered to each animal was 2 μg per 1-mg powder that wasreconstituted in water and delivered by IP injection. Serum samples werecollected 4 weeks after prime and two weeks after boost. Serumantibodies were determined using the standard ELISA and the results aresummarized in FIG. 13.

[0295] As can be seen in FIG. 13, compared to the untreated liquidvaccine (Control), the FD HBsAg vaccine composition (Group 1) showeddiminished immunogenicity. In addition, the particle size of thealum-containing powder had a pronounced effect on immunogenicity ofAlum-HBsAg. The immunogenicity of the freeze-dried formulations had aninverse correlation with the size of the particles (Groups 2, 3, and 4).The larger particle size fractions were less immunogenic than thesmaller particle size fraction. This is consistent with the SDS-PAGEresult and might be explained by the availability of HBsAg from thecoagulated alum matrix. Smaller particles have a greater specificsurface area, thereby allowing more HBsAg to be released from the alummatrix in vivo. An alternative explanation is that large coagulatedparticles are too big to be phagocytosed by antigen presenting cells,thus, the adsorbed vaccine antigen (HBsAg) is not available to theimmune system. Regardless of the mechanism, this data clearly indicatedthat large size particles associated with coagulation also correlatedwith the loss of vaccine potency.

[0296] The SFD formulations (Q and R) elicited a significantly higherantibody response than the FD counterparts. This result confirms thatalum coagulation caused an immunogenicity loss of HBsAg and that thefast freezing rate by SFD is an effective approach to preserving thealum adjuvant activity. The effect of alum concentration in the SFDpowder formulation on immunogenicity is important. Although no clearlydetectable coagulation was seen with SFD formulation Q, which contained3.0 w/v % of Alum HBsAg, this formulation induced an antibody titer thatwas approximately 1-log of magnitude lower than the IM injectioncontrol. The SFD formulation with 0.6% alum content had no coagulationand induced an antibody titer that was indistinguishable from the IMinjected animals (p>0.05, Student test). This result suggests thatlowering alum concentration and fast freezing are the most effectiveformulation parameters in minimizing alum particle coagulation, thus,maximizing the immunogenicity of the vaccine.

[0297] 3.5.2 EPI with Powdered Alum-DT

[0298] A commercial DT vaccine was used to illustrate the effect of theSFD process on an alum phosphate adjuvant-containing vaccinecomposition. The Alum-DT vaccine was dried by either conventional spraydrying (“SD”) or SFD, and the dried powder was then used to immunizehairless guinea pigs by using EPI. EPI delivers dry powder directly intothe epidermal layer where abundant antigen presenting cells (APCs) canbe activated to phagocytose or endocytose the dissolved antigen. Thestudy design of the guinea pig study is shown below in Table 3.4. TABLE3.4 In vivo immunogenicity study for Alum-DT powder formulations. GroupPowder dT & tT dose/ (n = 8) Formulation Formation Particle size mgpowder 1 S SD 38-53 μm 1.5 Lf/0.5 mg powder 2 T SFD 38-53 μm 1.5 Lf/0.7mg powder 3 Control Liquid N/A 1.5 Lf formulation

[0299] The formulations used in the study were as follows. FormulationS: alum phosphate (5 w/v %)/trehalose (5 w/v %). Formulation T: alumphosphate (1.5 w/v %)/trehalose (1.5 w/v %)/glycine (0.4 w/v %)/dextran(0.6 w/v %). Control: the commercial Alum-HBsAg product (Rhein Biotech)used as supplied by the manufacturer.

[0300] Formulation S (trehalose-based) was spray-dried using alaboratory-scale spray dryer (Mobile Minor, Niro, Inc). Formulation T,based on the combination of trehalose, glycine, and dextran, wasproduced using the SFD method of the present invention. For both powderformulations (Formulations S and T), the dried powder was subjected to aCIGIS technique (sieved to 38-53 μm size fraction) in order to match thesize of the SD powders. A dose of 1.5 Lf for both dT and tT was used,which is equivalent to approximately 0.5-mg of the SD powders and 0.7-mgof the SFD powders based on total protein analysis.

[0301] Further evaluation of alum coagulation by optical microscopy andparticle size analysis revealed the same findings that alum particleswere highly coagulated in the SD powder whereas the SFD powders yieldedmore gel-like suspensions upon rehydration. Serum samples were collected4 weeks after prime and two weeks post boost. Serum antibodies weredetermined using the standard anti-dT and tT ELISA. The results aresummarized in FIGS. 14a (anti-dT response) and 14 b (anti-tT response).It is apparent that the SD formulation elicited either no (for dT, seeFIG. 14a) or weak (for tT, see FIG. 14b) antibody responses. Incontrast, however, the SFD formulation elicited significantly higherresponses that were substantially equivalent to the responses induced bythe untreated liquid vaccine (Control) that was injectedintramuscularly.

EXAMPLE 4 Optimization of Alum-Adjuvanted Vaccine Compositions Preparedby SFD

[0302] 4.1 Objectives:

[0303] To optimize the performance of SFD alum-adjuvanted vaccinepowders and enhance the safety profile of the product by reducing alumcontent in the final composition, and to particularly address thefollowing issues: (a) further reduction of gel coagulation; (b)reduction in local tolerability issues associated with alum adjuvants;and (c) increase in vivo potency of the vaccine composition whenadministered by EPI.

[0304] 4.2 Materials:

[0305] The chemicals and excipients that were used in this study aresummarized below in Table 4.1. All alum formulations were concentratedto a desired concentration by centrifugation (Allergra 6R centrifuge,Beckman Instrument, Palo Alto, Calif.) prior to use. TABLE 4.1Chemicals/excipients used in the study. Chemical Lot # Source CommentAluminum phosphate 8934 Accurate Chemical Manufactured (Adjus-Phos, 2%and Scientific by HCI AlPO₄) (Westbury, NJ) Biosector (Frederikssund,Denmark) Aluminum hydroxide Accurate Chemical Manufactured (Alhydrogel,3% and Scientific by Superflos Al(OH)₃) Biosector (Vedbaek, Denmark)Diphtheria toxoid G9334 Accurate Chemical Manufactured (dT, MW 58 kDa)and Scientific by Statens Serum Institute, Denmark, and provided at 5mg/mL (1 Lf = 2.42 μg), used as supplied Alum phosphate- CSL LimitedBulk adjuvanted DT (Parkville, containing 5 Australia) w/v % alumphosphate adsorbed with both dT and tT at 563 Lf/mL Alum hydroxide-Rhein Amaericana 20 μg HBsAg adjuvanted hepatitis- S.A. adsorbed to 0.5B surface antigen (Buenos Ares, mg of (HBsAg) Argentina) aluminum or1.5-mg of aluminum hydroxide Dextran (MW 10,000 18H0568 Sigma Reagentgrade Da) (St. Louis, MO) Glycine 28H0103 Sigma Reagent grade Sodiumlauryl sulfate 17H0459 Sigma Sodium dodecyl sulfate (SDS); MW = 288TDalton Mannitol 127H0960 Sigma Reagent grade Pluronic F68 MPCS612B BASFPoloxamer 188NF; (Mount Olive, NJ) MW = 9,000 Trehalose dihydrate28H3797 Sigma Reagent grade Triton X-100 67H0044 Sigma t- Octylphenoxy-polyethyethanol; MW = 625 Polysorbate 80 18H5229 Sigma Polyoxy-ethylene- sorbitan monooleate; MW = 1,310

[0306] 4.3 Methods:

[0307] In general, placebo aluminum gels were formulated with a varietyof pharmaceutical excipients and dehydrated by SFD. After drying, thedry powder was reconstituted and examined under optical microscopy tomonitor gel coagulation. The degree of coagulation was also determinedby the sedimentation rate of the gel. An optimized formulation processwas then applied to prepare alum phosphate-adjuvanted hepatitis-Bsurface antigen (AlPO₄-HBsAg) where the alum content was {fraction(1/20)} of the commercial product. The amount of the antigen wasmeasured in vitro by SDS-PAGE or a micro BCA protein assay, and in vivopotency of the antigen was determined by intramuscular needle/syringeinjection and EPI of mice. The local tolerability of SFD alum adsorbeddiphtheria-tetanus toxoids (DT) vaccine administered by EPI was assessedin pigs and compared to intradermal (ID) injection.

[0308] 4.3.1 Spray-Freezing-Drying (SFD

[0309] The liquid formulation was delivered by the peristaltic pump(Model #77120-70, MasterFlex C/L, Barnant Company, Barrington, Ill.) ata flow rate of 2.0 mL/min into the ultrasonic atomizing system(Sono-Tek-Corporation, Milton, N.Y.) which consists of a spraying nozzle(Model #05793) and a power supply (Model #06-05108). The nozzle isequipped with a quasi-electric quartz crystal capable of vibrating at aspecific frequency that determines the size of the droplets. Thefrequency of 60 kHz spraying nozzle produces droplets mostly within therange of 20-80 μm. Atomized droplets were sprayed into the liquidN₂-containing pan (16-cm in diameter by 6-cm in height). Forformulations subjected to the spray-freezing/thawing experiment, thefrozen powder was transferred to a glass vial and thawed under ambientconditions. For frozen droplets undergoing drying, the pan containingfrozen particles in liquid nitrogen was transferred to a pre-cooled(−55° C.) shelf freeze dryer (Model #TDS2C2B5200, Dura-Stop, FTS System,Stone Ridge, N.Y.). Liquid nitrogen evaporated in a few minutes. Thefreeze-drying condition was set at −25° C. for 18 hours and 20° C. for10 hours. The ramping rate was 1° C./minute consistently. The vacuumpressure was 100 mT throughout the cycle. At the end of drying, thepowder-containing pans were transferred into a dry box purged withnitrogen (at <30% relative humidity) for powder collection. The samelyophilization cycle was used for freeze drying the liquid formulationsas the freeze-dried samples.

[0310] 4.3.2 Powder Formation by C/G/S Technique

[0311] To prepare powders of high density for EPI, some SFD formulationswere compressed in a stainless steel dye of 13-mm in diameter (CarverPress, Wabash, Ind.) at a pressure of 12,000-15,000 pounds for 5-10minutes. The compressed discs were ground manually using a mortar andpestle, and then the ground powder was manually sieved into the sizefraction of 53-75 μm using 3-inch sieves (Fisher Scientific Products,Pittsburgh, Pa.).

[0312] 4.3.3 Optical Microscopy

[0313] Visual analysis of the particles was performed using an opticalmicroscope (Model DMR, Leica, Germany) with 10×-eyepiece lens and10×-objective lens. The system was equipped with a Polaroid camerasystem for image output.

[0314] 4.3.4 Scanning Electron Microscopy

[0315] The external morphology of particles was examined using an Amray1810T scanning electron microscope (Amray, Bedford, Mass.). The powdersample was first sputtered coated with gold using a Hummer JR Technicsunit (Pergamon Corporation, King of Prussia, Pa.).

[0316] 4.3.5 Particle Size Analysis

[0317] The mean geometric/ aerodynamic diameter of particles in thevolume distribution was determined using a time-of-flight particle sizeanalyzer (Aerosizer, API, Minneapolis, Minn.). The mean volumetric sizewas calculated using the equipment software and applying a density of1.0 and such that the particle population between 10% (D₁₀) and 90%(D₉₀) could be determined for particle size distribution. Each analysisrequired approximately 3-5 mg of the powder sample.

[0318] 4.3.6 X-ray Powder Diffraction (XRD)

[0319] XRD measurement was conducted using a 35kV×15 mA Rigaku (D/max-β,CuKα radiation) X-ray diffractometer at room temperature and ambienthumidity. Samples were scanned at 0.1 degrees/second with 1 second counttime per increment. The range scanned was from 5 to 40 degrees.

[0320] 4.3.7 Alum Gel Coagulation Analysis

[0321] Two methods were used to determine alum gel coagulation, opticalmicroscopy and gel sedimentation. The powder sample was firstreconstituted in water to a concentration of 100 mg/mL withoutagitation. The gel solution was pipetted on a glass slide and examinedunder an optical microscope (Model DMR, Leica, Germany). The same gelsolution was then loaded in a 15-mL polystyrene conical tube (Falcon,Becton Dickinson, Franlkin Lakes, N.J.) and the sedimentation rate ofthe alum gel was monitored.

[0322] 4.3.8 HBsAg Adsorption to Alum Phosphate

[0323] To prepare 500 vaccine doses where each dose contains 20 μg HBsAgadsorbed onto 25 μg AlPO₄ in 100 μL, 8.9 mL of HBsAg antigen (1.4 mg/mL,pH 5.5) was mixed with 2.83 mL of AlPO₄ adjuvant (Adju-Phos, 2% AlPO₄),25 mL of 1.8% NaCl, and 13.3 mL of double-distilled water. The mixturewas inverted 5 times and then gently stirred overnight at roomtemperature (RT). The pH was then raised to 7.2 and the mixture wascentrifuged at 4,500 rpm for 20 min at RT. The supernatant was decantedand the pellet was re-suspended in 0.9% normal saline solution. Theamount of protein in the supernatant was determined by BCA proteinassay.

[0324] 4.3.9 SDS-PAGE

[0325] Coomassie colloidal-stained SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) was performed on a Nu-PAGE gel from Novex(San Diego, Calif.) (4-12% MES, running buffer, sample buffer, and/orDithiothreitol reducing agent). The alum- adjuvanted powder formulationswas reconstituted in water and centrifuged to remove the supernatant.The alum pellet was re-suspended in 200 mM sodium phosphate, pH 7 with0.1% SDS. The liquid suspension was then mixed with sample buffer fromthe Novex gel kit. The cocktail samples were then heated at 95° C. for 5minutes and vortexed prior to loading on the gel. The gels were run for35 minutes at 200V/120 b mA/25W using a power supply (PowerEase 500,Novex), and then coomassie stained (Novex Colloidal Blue Stain) anddestained with water. The gel images were scanned on a gel scanner(Model GS-700 Imaging Densitometer, BioRad) equipped with a quantitationsoftware (Quantity One), which can quantify the intensity of the gelbands. The unit of signal intensity is Optical Density (O.D.). Allsamples were compared against a molecular weight marker (Mark 12,Novex).

[0326] 4.3.10 Immunization and Serum Collection

[0327] Hairless guinea pigs (Charles River, Wilmington, Mass.) were usedto assess the immunogenicity of powder formulations following epidermalpowder inejction as described above in Example 3. In general, the methodof immunization was as follows. One mg of powder was dispensed into atrilaminate cassette. The cassette was inserted into the needlelesssyringe delivery device at the time of immunization. The device wasplaced against the left inguinal skin of the animals and actuated byreleasing the compressed helium at 40-bar pressure from the gascylinder. Control animals were immunized with 0.20 mL of liquid vaccinein saline by intramuscular (IM) injection using a 26½-gauge needle.Blood was collected via the kerotid blood vessel prior to eachvaccination and two weeks post boost.

[0328] 4.3.11 Local Reactogenicity Test

[0329] Pigs were anesthetized with a 1:1 mixture of Rompun and Telazol.The abdomen of each pig was tattooed on either side of the shot site.Each site received 2-mg powder formulation by EPI. Control sitesreceived an intra-dermal (ID) injection of reconstituted powders orunprocessed liquid vaccine. After immunization, each injection site wasinspected by palpation weekly.

[0330] Skin biopsies from the immunization sites were excised on day 42,fixed with 10% formalin, and embedded with paraffin. Sections of 6-μmthickness were cut, stained with hematoxylin and eosin (H&E), andvisualized under a light microscope (Nikon, Melville, N.Y.).

[0331] 4.3.12 ELISA

[0332] The antibody response to HBsAg was determined using a modifiedELISA method (see, e.g., Chen et al. (2000) Nature Med 6:1187-1190). Inparticular a 96-well plate (Costar, Fisher Scientific Products,Pittsburg, Pa.) was coated with 0.1 μg of antigen in 30 mM phosphatebuffered saline (PBS), pH 7.4, per well and allowed to sit overnight at4° C. Plates were washed 3 times with tris-buffered saline (TBS), pH7.4, containing 0.1% Brij-35, and incubated with test sera diluted inPBS containing 5% dry milk for 1.5 hour. A standard serum, containing aknown level of antibodies to HBsAg, was added to each plate and used tostandardize the titer in the final data analysis. The plates were thenwashed and incubated with biotin-labeled goat antibodies specific formouse immunoglobulin IgG or IgG subclasses (1:8,000 in PBS, SouthernBiotechnology Associate, Birmingham, Ala.) for 1 hour at roomtemperature. Following three additional washes, plates were incubatedwith streptavidin-horseradish peroxidase conjugates (SouthernBiotechnology) for 1 hour at room temperature. Finally, plates werewashed and developed with TMB substrate (Bio-Rad Laboratories, Melville,N.Y.). The endpoint titers of the sera were determined by 4-parameteranalysis using the Softmax Pro 4.1 program (Molecular Devices,Sunnyvale, Calif.) and defined as the reciprocal of the highest serumdilution with an OD reading above the background by 0.1. A referenceserum with a pre-determined titer was used on every plate to calibratethe titers and adjust assay-to-assay and plate-to-plate variation.

[0333] 4.4 In Vivo Performance, Enhanced Safety:

[0334] The following study was carried out to demonstrate the improvedsafety of alum-adjuvanted vaccines prepared using the SFD methods of thepresent invention. In particular, granuloma formation was assessed.Granuloma formation is the most common side effect associated withalum-adjuvanted vaccines administered intradermally or subcutaneously.

[0335] 4.4.1 Local Reactogenicity

[0336] The local reactogenicity to SFD alum-adjuvanted dT vaccinedeliverd using EPI was examined and compared with that of ID injection.The domestic white pig was chosen as an animal model for this testbecause it's epidermis is structurally similar to that of the human.Both EPI and ID injection with alum-adsorbed dT caused an erythemaresponse (localized skin reaction). The size of the erythema area wasobserved to be larger and more intense for the EPI administrations. Inall cases, the erythema completely resolved within 48 hours. The site ofthe EPI administrations appeared yellowish for an additional 2-3 days,but then restored to its normal color. becoming visuallyindistinguishable from normal skin 7 days post treatment. The studymatrix and the results from the reactogenicity study are reported belowin Table 4.2. TABLE 4.2 Granuloma formation following administration ofAlum-dT¹ Granuloma sites out of a total ten sites Formulation Alum RouteD7 D14 D21 D28 D35 D42 liquid Al(OH)3 ID 10 8 8 7 7 7 liquid AlPO4 ID 1010 10 10 8 8 Powder A² Al(OH)3 EPI 0 0 0 0 0 0 Powder B² AlPO4 EPI 0 0 00 0 0

[0337]¹ Each site was treated with 500 μg of alum-absorbed dT (Alum-dT)by ID injection of liquid vaccine or EPI of SFD powdered vaccine on day0. Granuloma formation was initially determined by weekly palpation for6 weeks, and then confirmed by histology on day 42.

[0338]²Powder A and Powder B were produced by formulating the respectivealuminum salt-adsorbed dT with 50% trehalose followed by a combined SFDand C/G/S process to produce 38-53 μm particles.

[0339] As can be seen, EPI administration of powdered compositionscontaining alum hydroxide or alum phosphate adjuvant did not result ingranulomas as determined by visual examination (Table 4.2). In contrast,most of the ID injection sites had a solid lump that could be detectedby palpation starting from day 7 and until the end of the study. At theend of the study (42 days post treatment), the sizes of the lumps in theanimals receiving the ID liquid vaccine injections ranged in 0.3 to 0.5mm in diameter and 0.2-0.3 mm in thickness.

[0340] The results of a histological examination of the EPI sites 42days after treatment are depicted in FIGS. 15a-15 e. In particular,histological examination showed a skin structure (see FIG. 15b) andcellular composition (FIG. 15c) that resembled those of normal skin (seeFIG. 15a). The lumps from the ID injection sites were found to betypical granulomas, composed of inflammatory cells with a connectivetissue capsule (FIG. 15d). The inflammatory cells were surrounded by acapsule of connective tissue that had completely replaced the normaltissue, again characteristic of granuloma formation. A closerexamination showed that the main class of infiltrating cells weremacrophages (see FIG. 15e). Both the alum hydroxide and alum phosphateliquid vaccine compositions induced granulomas when introduced by IDinjection. These results demonstrate that EPI administration of thepowdered vaccine compositions of the present invention provides a uniquesafety advantage over conventional administration routes.

[0341] 4.5 Optimization of the Powder Formulation:

[0342] Formulation parameters affecting the stability of the alum gelwere evaluated and optimized to produce SFD particles with minimal alumgel coagulation and superior particle characteristics suitable forepidermal powder injection techniques. In this regard, the SFD processhad already been adjusted to achieve the greatest freeze rate possibleby atomizing alum-containing liquid formulation into liquid nitrogen,thereby minimizing large-scale alum coagulation from occurring. In thisstudy, the enhancing effects of various excipient combinations wereinvestigated.

[0343] 4.5.1 Effect of Excipient Composition

[0344] High particle density and appropriate particle size are twoimportant powder properties that must be optimized for use intransdermal particle administrations such as EPI. It was expected thatexcipient composition would have a quantifiable influence on the densityof the SFD powder, since voids left behind in the particles byevaporation of ice crystals may cave-in during drying. Accordingly, aseries of alum hydroxide and alum phosphate compositions stabilized withvarious weight combinations of trehalose, mannitol and dextranexcipients were prepared and then assessed for gel performance andparticle characteristics. SFD particles were initially visually assessedusing SEM, and the results from the study are depicted in FIGS. 16a and16 b. In particular, the combination oftrehalose/mannitol/dextran=30%/30%/40% and a total solids content of 35w/w % resulted in particles with a corrugated morphology. SEMdemonstrated such morphology for the particles containing aluminiumhydroxide at 36 μg/1-mg powder (FIG. 16a) and for these containingaluminium phosphate at 50 μg/1-mg powder (FIG. 16b), where the particlesappear to have shrunk and thus become densified during the dryingprocess.

[0345] Next, alum coagulation was evaluated using optical microscopy onreconstituted powders that had been prepared by either a FD or SFDprocess. The results are depicted in FIGS. 17a-17 d, wherein FIG. 17ashows the FD alum hydroxide composition, FIG. 17c shows the FD alumphosphate composition, FIG. 17b shows the SFD alum hydroxidecomposition, and FIG. 17d shows the SFD alum phosphate composition. Ascan be seen, both FD formulations were highly coagulated when comparedto their SFD counterparts. Sedimentation experiments confirmed that theFD suspensions had settled, i.e., the top solution became clear, in oneminute but the SFD solutions took approximately 5 hours to clear. Whenthe composition of trehalose/mannitol/dextran excipeint was varied, thedensity of the corresponding SFD powder also changed. However, thedifference in coagulation was not distinguishable under opticalmicroscopy. Interestingly, the reconstituted gel settled at faster ratesas the content of mannitol was decreased. Sedimentation of aluminumphosphate (50 μg/1-mg powder) formulated in trehalose/mannitol/dextranat 40%/20%/40% completed, for example, in approximately 2 hours.

[0346] 4.4.2 Effect of Surface-Active Agents

[0347] The stabilizing effect of four common surface-active agentexcipients on the SFD alum-containing particles of the present inventionwere assessed. In particular, polysorbate 80 (786 Å, MW=1310), sodiumlauryl sulfate (SDS), (53 Å, MW=288), Pluronic F68, a block copolymer ofpolyoxyethylene and polyoxyproplyene (MW=9,000), and Triton X-100,octylphenol ethylene oxide condensate, average MW=625 were selected forthe study. All four surfactants were formulated withtrehalose/mannitol/dextran excipeints (present at 30%/30%/40%) at 3different concentrations, 0.5, 2, and 5% of the solid. Upon examinationof each reconstituted suspension solution by optical microscopy andsedimentation, no significant differences in alum coagulation among thevarious surface-active agents were observed. However, powders containingthe polysorbate 80, SDS, and Triton X-100 agents showed deterioratedpowder flowability, particularly at higher concentrations.

[0348] 4.4.3 Critical Concentration of Aluminum Salt

[0349] Although alum-adsorbed vaccines of low alum concentrations (<0.1w/v %) have reportedly been freeze-dried with preserved immunogenicity(see, e.g., U.S. Pat. No. 4,578,270 and European Patent No. 0130619 B1),these formulations are impractical considering that most commercialalum-adjuvanted vaccines are administered at a much higher alumconcentration, typically about 2-3 w/v %. However, as shown in Example 3above, the specific formulations described in U.S. Pat. No. 4,578,270and European Patent No. 0130619 B1 fail to stabilize alum salt fromcoagulation when freeze-dried at alum salt concentrations higher than1%. Therefore, alum salt concentrations might play a critical role inalum coagulation. Our hypothesis is that alum salt coagulation might beunavoidable when the alum concentration exceeds a critical level,despite the effectiveness of fast freezing by SFD. To identify thiscritical concentration, numerous formulations of aluminum phosphate andhydroxide were prepared by SFD at different alum salt concentrationsbefore SFD, excipient compositions, total solid concentrations, andpayloads. Some of these formulations are summarized below in Table 4.3.TABLE 4.3 Effect of alum concentration of the stability of SFDformulations¹ Total solid Liquid Alum Alum content in content priorConc. Prior to SFD powder to SFD Sample SFD (mg/mL) (μg/mg powder) (w/w%) Coagulation A 26.5 76.0 35 no B 30.3 76.0 40 no C 34.0 114.0 30 yes D28.4 114.0 25 no E 20.1 50.3 40 no F² 11.5 28.7 40 no

[0350] After SFD, the various sample formulations were assessed usingoptical microscopy to determine degree of alum gel coagulation. Resultsfrom the study are depicted in FIGS. 18a-18 d. As a result of theassessment, it was found that concentration of alum salt in the liquidformulation prior to SFD appears to be the critical factor. In thisregard, in the range of 26 to 34 mg/mL of alum salt in the liquid(starting) formulation, there is an obvious transition of alum gel intocoagulated form. Optical microscopy on certain reconstituted powderformulations (the samples reported Table 4.3), revealed the following.Formulation A (26.5 mg/mL) was free from coagulation (see FIG. 18a).Some coagulated alum particles were observed in Formulation B at 30.3mg/mL (see FIG. 18b). However, as the concentration of alum in thestarting liquid was further increased to 34.0 mg/mL (Formulation C),alum coagulation became much more pronounced (see FIG. 18c). Alumcoagulation had no direct correlation with the quantity of alum salt inthe powder, as Formulations C. and D have the same alum content (114μg/mg powder) and Formulation D was seen to be coagulation-free (seeFIG. 18d). This is because Formulation D was prepared from the liquidsuspension of alum salt at 28.4 mg/mL. Therefore, the critical alum saltconcentration for the SFD process appeared to be 30 mg/mL in the liquidgel. A possible explanation for this is that the high-concentration alumgel forms a dense matrix and affects the pattern of ice formation, whereice crystals are less likely to push freeze concentrate into smallcompartments compared to the case where the alum gel concentration islower. In addition, alum coagulation showed no correlation with thetotal solid content in the liquid gel prior to SFD (see Table 4.3). Ashigher total solid contents lead to more dense SFD particles, a totalsolid content of 35% or higher is preferred for producing powders foruse in EPI.

[0351] 4.6 In vivo Performance of the SFD Vaccine Compositions:

[0352] Based upon the above optimization assessment, it was decided thatthe SFD formulation should optimally start with a liquid formulationcontaining at least about 30 mg/mL alum gel concentration and have a 35%total solid content. SFD of one human dose of the commercial alumadsorbed HBsAg vaccine, which contains 500 μg of alum adjuvant, willyield approximately 28 mg of dried powder. Thus, to reduce the dry mass,the amount of alum in the vaccine dose must be reduced. As indicated bythe tolerability studies also described herein above, reduction in thealum in the vaccine should provide the added benefit of improving thetolerability of the SFD vaccine compositions. Accordingly, SFD powdervaccine compositions were made using 20 μg of the HBsAg antigen adsorbedto 25 μg alum phosphate adjuvant, and the immunogenicity of thisformulation was determined using either EPI or IM injection (afterreconstitution) of the SFD vaccine compositions and compared against thecommercial vaccine that contains the same HBsAg dose, but 20 times morealum adjuvant.

[0353] 4.6.1 Immunogenicity Studies

[0354] We first compared in hairless guinea pigs the immunogenicity ofpowdered formulations of HBsAg adjuvanted with either alum phosphate oralum hydroxide at a HBsAg:alum ratio of 1:25), denoted as Formulations Eand F (Table 4.3), respectively. These formulations were administered byeither EPI or IM injection of a liquid after the SFD particles werereconstituted. The results of the study are depicted in FIG. 19a. As canbe seen, all groups showed a significant increase in the antibodyresponse after the first and boost vaccinations (6 and 9 weeks afterprime respectively) were administered (see FIG. 19a). The antibodytiters in the animals vaccinated with the reconstituted powders wereequivalent to that induced by the untreated liquid formulation vaccine(P>0.05), suggesting the suitability of SFD for stabilization of dryalum formulation. All animals vaccinated by EPI developed significantantibody titers. Interestingly, the two alum salts appear to be equallyimmunogenic when delivered as dry powders in the skin.

[0355] In a subsequent study, we evaluated the immunogenicity of the drypowder formulation containing AlPO₄ at 1/20 of the regular alum content,i.e. 20μg of HBsAg adsorbed to 25 μg of aluminium. Because this highdose of vaccine can overwhelm the immune response in guinea pigs, thesubjects were vaccinated with only {fraction (1/10)} of the dose (2.0 μgHBsAg /2.5 μg of ALPO₄). The results of the study are depicted in FIG.19b, where it can be seen that the SFD formulation was efficient ineliciting antibody responses by IM injection as a reconstituted powder.At 2 weeks post-boost, the highest antibody titers were detected inanimals that received the SFD-reconstituted formulation with a geometricmean titer (GMT) of 5.6 (log 10). These titers were similar to thosedetected in animals that received the untreated liquid vaccineformulation (GMT=5.5 log 10) which contained twenty times more alum inthe form of alum hydroxide, suggesting that alum dose in the commercialvaccine can be reduced without compromising the efficacy. Thissignificantly reduced alum dose adds additional safety features toalum-adjuvanted vaccines delivered by EPI.

[0356] Accordingly, novel spray freeze-dried powder compositions andmethods for producing these compositions have been described. Althoughpreferred embodiments of the subject invention have been described insome detail, it is understood that obvious variations can be madewithout departing from the spirit and the scope of the invention asdefined by the appended claims.

What is claimed is:
 1. A process for the preparation of a powder, whichprocess comprises the step of spray freeze-drying an aqueous solution orsuspension comprising a pharmaceutical agent, said solution orsuspension having a solids content of 20% by weight or more.
 2. Aprocess according to claim 1, wherein the solution or suspension has asolids content of 30% by weight or more.
 3. A process according to claim2, wherein the solids content is 40% by weight or more.
 4. A processaccording to claim 1, wherein the pharmaceutical composition is anantigen.
 5. A process according to claim 4, wherein the antigen isadsorbed in an aluminum salt or calcium salt adjuvant.
 6. A processaccording to claim 4, wherein the antigen is a bacterial or viralantigen.
 7. A process according to claim 1, wherein the solution orsuspension further comprises (a) an amorphous excipient selected fromthe group consisting of monosaccharides, disaccharides, oligosaccharidesand polysaccharides; and (b) a crystalline excipient selected from thegroup consisting of carbohydrates, sugars and sugar alcohols.
 8. Aprocess according to claim 1, wherein the solution or suspension furthercomprises (a) an amorphous excipient selected from the group consistingof dextrose, sucrose, lactose, trehalose, cellobiose, raffinose,isomaltose and cyclodextrins, and (b) mannitol as a crystallineexcipient.
 9. A process according to claim 7, wherein the solution orsuspension further comprises (c) a polymer.
 10. A process according toclaim 9, wherein the polymer is dextran.
 11. A process according toclaim 7, wherein the solution or suspension further comprises (d) anamino acid or a physiologically acceptable salt thereof.
 12. A processaccording to claim 7, wherein the solution or suspension furthercomprises (c) a polymer and (d) an amino acid or physiologicallyacceptable salt thereof.
 13. A process according to claim 1, wherein thesolution or suspension further comprises trehalose, mannitol and dextranin a weight ratio of from about 3:3:4 to about 4:4:3.
 14. A processaccording to claim 1, wherein the solution or suspension is sprayed froman ultrasonic nozzle.
 15. A process according to claim 1, wherein thesolution or suspension is sprayed into liquid nitrogen.
 16. A processaccording to claim 1, wherein the solution or suspension is sprayed intoa liquified gas and the liquified gas containing the resulting frozendroplets of the solution or suspension is subjected to a two stagedrying process comprising: (i) a first drying stage which is performedat a temperature of from about −50° C. to 0° C. for a period of about 4to 24 hours under a pressure of about 20 to 500 mT; and (ii) a seconddrying stage which is performed at a temperature of from about 5 to 30°C. for a period of about 5 to 24 hours under a pressure of less than 100mT.
 17. A process according to claim 1, wherein the resulting sprayfreeze-dried particles are collected, washed and dried.
 18. A processaccording to claim 17, wherein the dried particles are sieved.